Pharmaceutical formulations for sustained release of sebacoyl dinalbuphine ester

ABSTRACT

The present invention relates to injectable, extended-release, pharmaceutical formulations comprising a nalbuphine ester prodrug homogenously dissolved in a solution comprising a pharmaceutically acceptable oil and an oil-miscible retaining solvent, as well as manufacturing processes and medical uses of the formulations. The invention further provides methods for adjusting the duration of action of the formulations by varying the ratio of the pharmaceutically acceptable oil and the oil-miscible retaining solvent.

This application is a continuation of U.S. patent application Ser. No.15/166,403, filed May 27, 2016, which is a continuation-in-part of U.S.patent application Ser. No. 14/723,996, filed May 28, 2015, and alsoclaims priority to U.S. Provisional Application No. 62/255,805, filedNov. 16, 2015. These references are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to pharmaceutical compositions.More particularly, the present invention relates to extended releaseformulations of nalbuphine ester prodrugs such as sebacoyl dinalbuphineester (“SDE”).

BACKGROUND OF THE INVENTION

Opioids have been widely regarded as the most effective drugs for thetreatment of pain, and the use of opioids in the management of acutesevere pain and chronic pain is considered the standard of care.Prescription opioids are available as immediate-release (IR) or extendedrelease (ER) formulations. Compared with IR formulations, ERformulations allow a controlled release of the active agent to provide aprolonged plasma drug level within the therapeutic window; ERformulations also provide a lower maximum concentration (C_(max)), fewerpeak-to-trough fluctuations, and less frequent dosing (J MultidiscipHealthc. 2013; 6: 265-280). However, the effective duration of most ofthe current market available extended release opioids is less than 3days. For example, nalbuphine is a short-acting drug with a duration ofaction of 3-5 hours after being administered via intravenous (IV),subcutaneous (SC), or intramuscular (IM) injection. As such, frequentinjections or administrations of nalbuphine are needed for patientssuffering from dramatic or long-lasting pain.

U.S. Pat. No. 6,197,344 discloses several controlled release suspensionformulations for subcutaneous administration, each of which comprisesthe opioid analgesic butorphanol in the form of microparticles having anaverage particle size of from about 5 to 25 microns. It is stated thatthe suspension formulations can be used to relieve pain for 12 to 24hours. Furthermore, because the particle sizes of butorphanolmicroparticles are too large, the suspensions are considered notsuitable for being administered by intramuscular injection or for beingsterilized by filtration.

U.S. Pat. No. 8,455,508 discloses an oil- and pH-controlledbuprenorphine-release formulation, which can be administered bysubcutaneous or intramuscular injection. The formulation is in the formof an emulsion and needs to be prepared through several sterilizingprocedures during its manufacturing process, which is time-consuming andnot cost effective in large scale production.

U.S. Pat. No. 6,225,321 discloses several extended release formulationsfor intramuscularly administrating nalbuphine ester prodrugs, e.g.,sebacoyl dinalbuphine ester (“SDE”). The formulations are prepared bymixing the nalbuphine ester prodrugs with therapeutically injectableoils and excipients (such as methyl paraben, propyl paraben, BHA, BHT,cremophore EL, pluronic, solutol, or span). It is stated that a singledose of the formulation could give an analgesic effect maintained for 4to 5 days when the injection volume is 7.15 mL. However, 5 mL wasreported for adults as the maximum volume for a single intramuscularinjection. Large-volume injections (3 mL or greater) are rarelyadministered clinically, and may cause serious injection siteirritation.

U.S. Pat. No. 6,703,398 discloses formulations for orally administratingnalbuphine or nalbuphine ester. The oral formulations are prepared bymixing nalbuphine or nalbuphine ester with an oily substance, and asolubility-assisting agent. It is stated that the solubility-assistingagent is used to improve bioavailability and half-life of nalbuphine ornalbuphine ester. However, the apparent half-life (t_(1/2)) ofnalbuphine from the oral formulation was only about 24 hours, and itwould require the dosing interval of the oral formulation to beapproximately every 8 to 12 hours to exert efficacy. Such a dosingfrequency is not practical or desirable for patients suffering fromlong-term or severe pain, for example, post-surgical pain.

Although the use of emulsion or oil-based vehicles in preparing extendedrelease formulations of opioids is not unknown, the complexity ofsterilization and the limited solubility of nalbuphine ester prodrugs inoily substances make it difficult to achieve extended releaseformulations which can release nalbuphine ester prodrugs in awell-controlled manner, can be administered to patients in a lowinjection volume, and can be prepared by a simple and cost-effectivemethod, applicable in industrial scale manufacturing. There is a need toprepare extended release formulations with predetermined release periodsby simple and cost effective methods.

BRIEF SUMMARY OF THE INVENTION

The following presents a simplified summary of the invention in order toprovide the reader a basic understanding. This summary is not anextensive overview of the invention. It is not intended to, and doesnot, delineate the scope of the present invention. Rather, its solepurpose is to present some concepts disclosed herein in a simplifiedform as a prelude to the more detailed description that is presentedlater.

In one aspect, the present disclosure provides pharmaceuticalformulations each comprising a nalbuphine ester prodrug and arelease-controlling solution, wherein the formulations are suitable foradministration by injection and release the nalbuphine ester prodrug inan extended manner. In some embodiments, the nalbuphine ester prodrug ishomogenously dissolved in the release-controlling solution. In someembodiments, the release-controlling solution comprises an oil-miscibleretaining solvent and a pharmaceutically acceptable oil.

The present disclosure also provides pharmaceutical formulations eachcomprising the nalbuphine ester prodrug dissolved in thepharmaceutically acceptable oil and the oil-miscible retaining solvent.In some embodiments, the concentration of the nalbuphine ester prodrugin the formulation is greater than the solubility of the nalbuphineester prodrug when added to a mixture of the pharmaceutically acceptableoil and the oil-miscible retaining solvent. In some embodiments, theweight ratio of the oil-miscible retaining solvent to thepharmaceutically acceptable oil is equal to or greater than about 1.1:1.Such pharmaceutical formulations may provide an extended release periodof the nalbuphine ester prodrug.

The inventors have discovered that by increasing the weight ratio of theoil-miscible retaining solvent to the pharmaceutically acceptable oil(the “retaining solvent/oil ratio”), the release period of thenalbuphine ester prodrug from the formulation may be extended. In someembodiments, the nalbuphine ester prodrug is sebacoyl dinalbuphine ester(SDE). The pharmaceutically acceptable oil may be a vegetable oil, forexample, sesame oil, cottonseed oil, castor oil or a mixture thereof.The oil-miscible retaining solvent may be benzyl benzoate, benzylalcohol, or a mixture thereof. In some embodiments, the pharmaceuticallyacceptable oil is sesame oil and the oil-miscible retaining solvent isbenzyl benzoate.

In some embodiments, the solubility of the prodrug, e.g., SDE, in theoil-miscible retaining solvent is equal to or greater than about 100mg/mL. For example, the solubility of SDE in the oil-miscible retainingsolvent may be equal to or greater than 300 mg/mL.

In some embodiments, the concentration of the prodrug, e.g., SDE, in theformulation is equal to or greater than about 70 mg/mL. For example, theconcentration of SDE in the formulation may be about 70-300 mg/mL,70-150 mg/mL, or about 70-100 mg/mL. In some embodiments, theconcentration of SDE in the formulation is about 75 mg/mL or about 80mg/mL.

In some embodiments, the retaining solvent/oil ratio is about 0.5 toabout 19. In some embodiments, the retaining solvent/oil ratio is equalto or greater than 1.1:1. For example, the retaining solvent/oil ratiomay be about 0.8-1.2:1, about 0.65-2:1, about 1-2:1, or about 1-3:1. Insome embodiments, the retaining solvent/oil ratio is about 1.12:1, about1.18:1, about 0.65:1, about 2:1 or about 3:1.

In some embodiments, the concentration of SDE in the formulation isabout 70-80 mg/mL, and the weight ratio of benzyl benzoate to sesame oilis about 1.1-1.2:1. In one embodiment, the concentration of SDE in theformulation is about 75 mg/mL, and the weight ratio of benzyl benzoateto sesame oil is about 1.12:1. In one embodiment, the concentration ofSDE in the formulation is about 80 mg/mL, and the weight ratio of benzylbenzoate to sesame oil is about 1.18:1.

In some embodiments, the concentration of the nalbuphine ester prodrugin the formulation is greater than the solubility of the nalbuphineester prodrug when added to the mixture of the pharmaceuticallyacceptable oil and the oil-miscible retaining solvent. For example, thenalbuphine ester prodrug may be SDE; the pharmaceutically acceptable oilmay be sesame oil and the oil-miscible retaining solvent may be benzylbenzoate; the weight ratio of benzyl benzoate to sesame oil may be about0.8-1.2:1; and the concentration of SDE in the formulation may begreater than about 70 mg/mL.

In some embodiments, the weight ratio of the oil-miscible retainingsolvent to the pharmaceutically acceptable oil is equal to or greaterthan about 1.1:1. For example, the nalbuphine ester prodrug may be SDE;the pharmaceutically acceptable oil may be sesame oil and theoil-miscible retaining solvent may be benzyl benzoate; the weight ratioof benzyl benzoate to sesame oil may be about 1.1-3:1; and theconcentration of SDE in the formulation may be greater than about 70mg/mL.

In some embodiments, the formulation is an extended or sustained releaseformulation. In some embodiments, the present formulation has a durationof action of equal to or greater than about 5 days, or about 6 days. Insome embodiments, the present formulation has a release period of equalto or greater than about 10 days, about 12 days, or about 14 days. Insome embodiments, the retaining solvent/oil ratio is greater than about1, and the duration of action of the pharmaceutical formulation is equalto or greater than about 5 days or about 6 days, and/or the releaseperiod of the pharmaceutical formulation is equal to or greater thanabout 10 days, about 12 days or about 14 days. In some embodiments, theretaining solvent/oil ratio is less than about 1, and the duration ofaction of the pharmaceutical formulation is less than about 6 days,and/or the release period of the pharmaceutical formulation is less thanabout 14 days.

The present formulations may be intramuscularly or subcutaneouslyadministered to a subject. In some embodiments, the present formulationis suitable for administration by intramuscular injection.

In some embodiments, the formulation further comprises adding asolubilizing agent and/or a neutralizing agent.

In another aspect, the present invention provides a method for preparingan extended release formulation comprising a nalbuphine ester prodrugwith a predetermined release period, comprising providing anoil-miscible retaining solvent and a pharmaceutically acceptable oil,wherein the weight ratio of the retaining solvent to the oil is adjustedbased on the predetermined release period; and mixing the nalbuphineester prodrug with the retaining solvent and the oil to give ahomogeneously dissolved solution. In some embodiments, the step ofmixing the prodrug with the retaining solvent and the oil comprisesmixing the prodrug with the oil-miscible retaining solvent to give aclear solution, and then mixing the clear solution with thepharmaceutically acceptable oil. In some embodiments, the step of mixingthe prodrug with the retaining solvent and the oil comprises mixing theprodrug with a mixture of the oil-miscible retaining solvent and thepharmaceutically acceptable oil.

In some embodiments, the predetermined release period is equal to orgreater than about 10 days, about 12 days or about 14 days, and theretaining solvent/oil ratio is adjusted to greater than about 1. Forexample, when a duration of action of equal to or greater than about 5or 6 days is intended for the present formulation, the retainingsolvent/oil ratio may be adjusted to greater than about 1. When aduration of action of equal to or greater than about 5 or 6 days, and/ora release period of equal to or greater than 14 days, is intended forthe present formulation, the retaining solvent/oil ratio may be adjustedto greater than about 1. In some embodiments, the predetermined releaseperiod is less than about 14 days, and the retaining solvent/oil ratiocan be adjusted to less than about 1. In some embodiments, when aduration of action of less than about 6 days, and/or a release period ofless than 14 days, is intended for the present formulation, theretaining solvent/oil ratio can be adjusted to less than about 1.

The present invention also provides a method for preparing apharmaceutical formulation, comprising dissolving the nalbuphine esterprodrug in the oil-miscible retaining solvent; and mixing the resultingsolution with the pharmaceutically acceptable oil to give a homogenoussolution, wherein the formulation is suitable for administration byinjection. In some embodiments, the nalbuphine ester prodrug isdissolved in the formulation at a concentration greater than thesolubility of the nalbuphine ester prodrug when added to a mixture ofthe oil-miscible retaining solvent and the pharmaceutically acceptableoil.

In some embodiments, the nalbuphine ester prodrug is SDE. In someembodiments, the solubility of the prodrug, e.g., SDE, in theoil-miscible retaining solvent is equal to or greater than about 100mg/mL. In some embodiments, the concentration of the prodrug, e.g., SDE,in the formulation is greater than about 70 mg/mL. In some embodiments,the pharmaceutically acceptable oil is soybean oil, peanut oil, sesameoil, or a mixture thereof; and the oil-miscible retaining solvent isbenzyl benzoate, benzyl alcohol, or a mixture thereof. For example, theoil may be sesame oil and the retaining solvent may be benzyl benzoate.In some embodiments, the weight ratio of benzyl benzoate to sesame oilis about 0.8-1.2:1. In some embodiments, the formulation is suitable foradministration by intramuscular or subcutaneous injection.

In some embodiments, the method can further comprise adding asolubilizing agent and/or a neutralizing agent.

In some embodiments, the methods of the invention can further includethe step of filtering the resulting homogeneous solution with a bacteriaproof filter.

In another aspect, the present invention provides a method of treatingpain, comprise administering a therapeutically effective amount of thepharmaceutical formulation of the invention to a subject in needthereof. In some embodiments, the formulation being administeredsubstantially consists of SDE, sesame oil and benzyl benzoate, whereinthe weight ratio of benzyl benzoate to sesame oil is about 0.8-1.2:1. Insome embodiments, the concentration of SDE in the formulation beingadministered is greater than about 70 mg/mL.

In some embodiments, the formulation is administered by intramuscularinjection. In some embodiments, the formulation is administered 6-36hours prior to the onset of pain symptoms. In some embodiments, theonset of pain symptoms is during or after a surgical operation on thesubject. In some embodiments, the formulation is administered to delivera total dose of up to about 160 mg of SDE.

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only, andare intended to provide further, non-limiting explanation of thedisclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the accumulated dissolution profiles of R1, AF3, AF1, AF4,R2, and N7 formulations.

FIG. 1B shows the accumulated dissolution profiles of N8 and N9formulations.

FIG. 2 shows the accumulated dissolution profiles of AF3, AF1 and AF4formulations.

FIG. 3 shows the plasma nalbuphine concentration in dogs afterintramuscular injection of AF3, AF1 and AF4 formulations (total dose:160 mg/dog).

FIG. 4 shows the log plasma nalbuphine concentration in dogs afterintramuscular injection of AF3, AF1 and AF4 formulations (total dose:160 mg/dog).

FIG. 5 shows the cumulative relative fraction absorbed (CRFA)-timeprofiles of nalbuphine in dogs after intramuscular injection of AF3, AF1and AF4 formulations (total dose: 160 mg/dog).

FIG. 6 shows the accumulated dissolution profiles of AF1 and F8formulations.

FIG. 7 shows the mean plasma concentration-time profile of nalbuphineafter human subjects received a single intramuscular injection of 17 mgNubain® (Nalbuphine-HCl) or escalating doses (75, 100, 125, and 150 mg)of the F8 formulation.

FIG. 8 shows the mean whole blood concentration-time profile ofnalbuphine after human subjects received a single intramuscularinjection of the F8 formulation (total dose: 150 mg/person).

FIG. 9 shows the distribution statistics of the consumption of oralketorolac through 48 hours after hemorrhoid operation on subjects whoreceived SDE prior to surgery or on subjects who received placebo priorto surgery.

FIG. 10 shows the plot of adjusted VAS scores over time through 0 hr to7 days after hemorrhoid operation of mITT population.

FIG. 11 shows the saturated concentration/solubility of SDE in differentsolvent systems containing different alkyl alcohols.

FIG. 12 shows the accumulated dissolution profiles of N1-N5formulations.

FIG. 13 shows the accumulated dissolution profiles of N5, N7 and N10formulations.

FIG. 14 shows the accumulated dissolution profiles of AF1, N2, and F8formulations.

FIG. 15 shows the accumulated dissolution profiles of N2, N4, N13, andN14 formulations.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description and examples illustrate certainembodiments of the present invention. Those of skill in the art willrecognize that there are numerous variations and modifications of thisdisclosure that are encompassed by its scope. Accordingly, thedescription of certain embodiments should not be deemed as limiting.

As used herein, the term “extended release” or a similar expression, forexample, “sustained release” means that an active pharmaceuticalingredient or prodrug thereof may be continuously released from a drugformulation or pharmaceutical formulation over an extended period oftime after being administered (e.g., 24 hours or longer, such as equalto or greater than 3 days, equal to or greater than 5 days, equal to orgreater than 6 days, equal to or greater than 7 days, or equal to orgreater than 14 days from dosing). Other related terms include “releasedin a sustained manner.”

As used herein, the term “release period” means a period of time duringwhich an active ingredient or prodrug thereof is available forabsorption and pharmacological effect (e.g., to treat pain) afteradministration.

As used herein, the term “duration of action” means the length of timean active ingredient or prodrug thereof exhibits a desired pharmacologiceffect, e.g., an analgesic effect, after administration. This isdetermined by the amount of time drug concentration is at or above aminimum effective concentration.

As used herein, the term “pharmaceutically acceptable oil” refers to anoil that may be used to prepare pharmaceutical formulations containingan active ingredient, without the oil causing an unacceptable adverseeffect. As used herein, the term “therapeutically injectable oil” refersto an oil that can be used to prepare pharmaceutical formulationscontaining an active ingredient, and the formulations later can beinjected into a patient for clinical or therapeutic use, without the oilcausing an unacceptable adverse effect. Thus, a “pharmaceuticallyacceptable oil” may also refer to a “therapeutically injectable oil.”

As used herein, the term “release-controlling solution” refers to asolution that may be used to regulate or control the release rate orrelease period of an active ingredient or prodrug thereof from aformulation.

As used herein, the term “oil-miscible retaining solvent” refers to anorganic solvent that is miscible with oil and may be used to slow therelease of an active ingredient or prodrug thereof in a formulation soas to modify the rate of drug delivery or to modify the solubility ofthe drug in the pharmaceutically acceptable oil. Dissolving the drug orpro-drug thereof in the oil-miscible retaining solvent before thepharmaceutically acceptable oil is added may allow for a concentrationof drug or prodrug thereof in the formulation that is higher than thesolubility of the drug or prodrug when it is added to a pre-made mixtureof the oil and the retaining solvent.

As used herein, the term “solubilizing agent” means a substance that maybe used to increase the solubility of an active ingredient or prodrugthereof in a liquid formulation, and is miscible with the liquidformulation.

As used herein, the term “neutralizing agent” means a substance that maybe used to neutralize an acid generated during or after administrationof a drug formulation.

As used herein, the word “dissolve” (e.g., as in fully dissolve) or“dissolved” means that a non-aqueous substance (e.g., a solid) becomesor causes to become incorporated into a liquid so as to form ahomogeneous solution.

As used herein, the term “homogenously dissolved” means that anon-liquid (e.g., a solid or amorphous) compound is completely dissolvedin a solvent, or a solvent system or mixture, to give a homogenoussolution which, for example, does not contain particles or precipitatesof the compound to be dissolved.

As used herein, the terms “suitable for injection” or “suitable foradministration by injection” mean that a pharmaceutical composition isin such a form or state that makes it clinically possible or ideal foradministration by injection, e.g., subcutaneous injection, intravenousinjection, or intramuscular injection.

As used herein, the term “solubility” means the maximum amount of asolute (e.g., an active ingredient or a prodrug thereof) that can bedissolved in a given amount of a solvent at a particular temperature.Unless otherwise specified, the solubilities presented herein are atroom temperature (e.g., 25-28° C.). The term “saturated concentration”refers to the concentration at which no more of a solute will dissolvein a solvent and additional amounts of solute will appear as a separatephase (e.g., as a precipitate).

As used herein, the singular forms “a”, “and”, and “the” are used hereinto include plural referents unless the context clearly dictatesotherwise.

As used herein, the term “about” means within an acceptable standarderror of the mean when considered by one of ordinary skill in the art.Other than in the operating/working examples, or unless otherwiseexpressly specified, all of the numerical ranges, amounts, values, andpercentages such as those for quantities of materials, durations oftimes, temperatures, operating conditions, ratios of amounts, orreflection angles disclosed herein should be understood as modified inall instances by the term “about.” In the context of the retainingsolvent/oil ratio, of the concentration of nalbuphine ester prodrug, orof time, for example, the amount of time for each step of the disclosedmethods, “about” as used herein indicates that the calculation or themeasurement of the value allows some slight imprecision without having asubstantial effect on the chemical or physical attributes of thedisclosed formulations or methods. If, for some reason, the imprecisionprovided by “about” is not otherwise understood in the art with thisordinary meaning, then “about” as used herein indicates a possiblevariation of up to 5% in the value.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of the ordinaryskill in the art to which this invention belongs.

Pharmaceutical Formulations

In one aspect, the present invention provides pharmaceuticalformulations each comprising a nalbuphine ester prodrug and arelease-controlling solution, wherein the formulations are suitable foradministration by injection and release the nalbuphine ester prodrug inan extended or sustained manner. In some embodiments, the nalbuphineester prodrug is homogenously dissolved in the release-controllingsolution. The release-controlling solution may comprise an oil-miscibleretaining solvent and a pharmaceutically acceptable oil.

The present disclosure also provides pharmaceutical formulations eachcomprising the nalbuphine ester prodrug dissolved in thepharmaceutically acceptable oil and the oil-miscible retaining solvent.In some embodiments, the concentration of the nalbuphine ester prodrugin the formulation is greater than the solubility of the nalbuphineester prodrug when added to a mixture of the pharmaceutically acceptableoil and the oil-miscible retaining solvent. In some embodiments, theweight ratio of the oil-miscible retaining solvent to thepharmaceutically acceptable oil is equal to or greater than about 1.1:1.Such pharmaceutical formulations may provide an extended release periodof the nalbuphine ester prodrug.

The nalbuphine ester prodrug may show a better solubility in an oilysubstance as compared with nalbuphine. For example, the prodrug may beany of the nalbuphine polyester derivatives disclosed in U.S. Pat. No.6,225,321. In some embodiments, the nalbuphine ester prodrug may besebacoyl dinalbuphine ester (“SDE”). In some embodiments, theconcentration of SDE in the formulation is about 70-300 mg/mL, about70-150 mg/mL, or about 70-100 mg/mL. For example, the concentration ofSDE in the formulation may be about 70 mg/mL, about 75 mg/mL, about 80mg/mL, about 100 mg/mL, or about 150 mg/mL.

In some embodiments, the solubility of the nalbuphine ester prodrug inthe retaining solvent is equal to or greater than about 100 mg/mL. Inother embodiments, the oil-miscible retaining solvent can dissolve thenalbuphine ester prodrug at a high concentration, e.g., more than about150 mg/mL, or more than about 300 mg/mL, and is miscible with thepharmaceutically acceptable oil. In some embodiments, the oil-miscibleretaining solvent is benzyl benzoate, benzyl alcohol, or any mixturethereof, and the nalbuphine ester prodrug is SDE. The solubilities ofSDE in benzyl benzoate and benzyl alcohol are greater than 300 mg/mL andgreater than 500 mg/mL, respectively.

The pharmaceutically acceptable oil may be a vegetable oil. In someembodiments, the pharmaceutically acceptable oil may be sesame oil,castor oil, cottonseed oil, soybean oil, corn oil, sunflower oil,arachis oil, olive oil, or any mixture thereof. In some embodiments, theoil is sesame oil, cottonseed oil, or castor oil. For example, the oilmay be sesame oil.

The oil-miscible retaining solvent may be an organic solvent that ismiscible with the pharmaceutically acceptable oil, for example, benzylbenzoate, benzyl alcohol, or a mixture thereof. In some embodiments, theretaining solvent is benzyl benzoate. For example, the pharmaceuticallyacceptable oil may be sesame oil and the oil-miscible retaining solventmay be benzyl benzoate.

The pharmaceutically acceptable oil is miscible with the retainingsolvent. When the formulation is administered into a subject bysubcutaneous or intramuscular injection, the oil and the retainingsolvent may form a matrix to retain the nalbuphine ester prodrug, so asto release the prodrug from the formulation in an extended/controlledmanner. When the weight ratio of the retaining solvent to the oil isincreased, the release period of the prodrug from the formulation may beextended or prolonged. Without wishing to limit the invention to anyparticular theory or mechanism of operation, it is believed that theoil-miscible retaining solvent serves as the key element in theformulation to retain the prodrug in the matrix.

As used herein, retaining solvent/oil ratios may be expressed as either,for example, 3:1 or 3. For example, a retaining solvent/oil ratioexpressed as 0.5:1 is the same as a retaining solvent/oil ratioexpressed as 0.5. A range of retaining solvent/oil ratios may beexpressed as 1:1-3:1, 1-3:1 or 1-3, for example.

By controlling/adjusting the retaining solvent/oil ratio, the presentformulations may regulate the release rate/release period of thenalbuphine ester prodrug from the formulation. For example, when theretaining solvent/oil ratio is adjusted to 3:1, the formulation may showa release period significantly longer than that of the formulation witha retaining solvent/oil ratio of 0.5:1. In some embodiments, when theretaining solvent/oil ratio is adjusted to about 0.65, about 99% of theprodrug is released from the formulation at 144 hours from dosing; whenthe retaining solvent/oil ratio is adjusted to about 1, about 90% of theprodrug is released from the formulation at 144 hours from dosing; whenthe retaining solvent/oil ratio is adjusted to about 2, about 80% of theprodrug is released from the formulation at 144 hours from dosing. Insome embodiments, when the retaining solvent/oil ratio is adjusted togreater than about 1, the present formulation may have a release periodof equal to or greater than 10 days, 12 days or 14 days. In someembodiments, when the retaining solvent/oil ratio is adjusted to greaterthan about 1, the present formulation may have a duration of action ofequal to or greater than 5 days or 6 days.

In some embodiments, when the retaining solvent/oil ratio is adjusted togreater than about 1, the present formulation may have a release periodof equal to or greater than 14 days or may have a duration of action ofequal to or greater than 6 days. In some embodiments, when the retainingsolvent/oil ratio is adjusted to about 1-3 or about 1-2, the presentformulation may have a duration of action of equal to or greater thanabout 5 or 6 days, and/or a release period of equal to or greater thanabout 10, 12 or 14 days. In some embodiments, when the retainingsolvent/oil ratio is adjusted to less than about 1, the presentformulation may have a release period of less than 14 days, and/or aduration of action of less than 6 days.

In some embodiments, the retaining solvent/oil ratio is about 0.5 toabout 19. In some embodiments, the retaining solvent/oil ratio is about0.65-8:1, about 0.65-3:1, about 0.65-2:1, about 1-8:1, about 1-3:1,about 1-2:1, or about 0.8-1.2:1. For example, the retaining solvent/oilratio may be about 0.5, 0.65, 0.8, 1, 1.12, 1.18, 1.2, 2, 3 or 8.

In some embodiments, the oil-miscible retaining solvent is benzylbenzoate, benzyl alcohol, or any mixture thereof; the nalbuphine esterprodrug is SDE; and the pharmaceutically acceptable oil is sesame oil,cottonseed oil, castor oil, or any mixture thereof. For example, theoil-miscible retaining solvent may be benzyl benzoate, the nalbuphineester prodrug may be SDE, and the oil may be sesame oil, where theweight ratio of benzyl benzoate to the oil (the “BB/oil ratio”) may beabout 0.5 to about 16. The present formulations when being prepared withthe BB/oil ratio of 16 may show a longer release period than as preparedwith the BB/oil ratio of 0.5. In some embodiments, the BB/oil ratio isabout 0.65-8:1, about 0.65-3:1, about 0.65-2:1, about 1-8:1, about1-3:1, about 1-2:1, or about 0.8-1.2:1. For example, the BB/oil ratiomay be about 0.5, 0.65, 0.8, 1, 1.12, 1.18, 1.2, 2, 3 or 8. Inembodiments where the BB/oil ratio is adjusted to about 1:12, thepresent formulation may have a release period of equal to or greaterthan about 10 days, 12 days or 14 days, and/or a duration of action ofequal to or greater than about 5 days or 6 days. In particular, whenBB/oil ratio is adjusted to about 1:12, the present formulation may havea release period of equal to or greater than about 14 days, and/or havea duration of action of equal to or greater than about 6 days.

In some embodiments, the oil-miscible retaining solvent is a mixture ofbenzyl benzoate and benzyl alcohol. In some embodiments, the combinedweight ratio of benzyl benzoate and benzyl alcohol to thepharmaceutically acceptable oil (“BB+BA/oil ratio”) may be 0.5-19, about0.82-19, about 0.5-16, about 0.65-8:1, about 0.65-3:1, about 1-8:1, orabout 1-3:1. In some embodiments, the BB+BA/oil ratio may be about0.82:1 or about 19:1.

In some embodiments, the present formulation comprises SDE, benzylbenzoate, and sesame oil, wherein the formulation is administered to asubject by intramuscular injection, the weight ratio of benzyl benzoateto sesame oil is greater than 1:1, the concentration of SDE in theformulation is greater than 70 mg/mL, and the duration of action of theformulation is equal to or greater than 6 days.

In some embodiments, the pharmaceutical formulations of the presentinvention are suitable for injection into a subject in need thereof. Itis known in the art that a formulation has to be a homogeneous solutionor a homogeneous suspension in order to be qualified as an injectableformulation. More particularly, an injectable suspension should containat most 0.5-5.0% solids and have an average particle size of less than 5micrometers in order to be used as a pharmaceutically acceptablesuspension for intramuscular injection (see R. M. Patel, ParenteralSuspension: an Overview, Int. J. Curr. Pharm. Res., 2010, 2(3):3-13). Insome embodiments, the formulations of the present disclosure arehomogeneous and stable solutions and, therefore, are suitable foradministration by intramuscular injection. In some embodiments, thenalbuphine ester prodrug is homogenously dissolved in the formulation.

It should be understood that, due to the difference between thesolubility of SDE in the retaining solvent and in the oil, thesolubility of SDE is higher in formulations with higher retainingsolvent/oil ratios. For example, the solubilities of SDE in benzylbenzoate and benzyl alcohol are greater than 300 mg/mL and greater than500 mg/mL, respectively, while the solubilities of SDE in sesame oil,castor oil, and cottonseed oil are about 6 mg/mL, about 13 mg/mL, andabout 6 mg/mL, respectively. Based on a series of solubility testsconducted by the inventors, it was demonstrated that when the weightratio of benzyl benzoate to sesame oil is about 1, the solubility (i.e.,the saturated concentration) of SDE when added to the mixture of benzylbenzoate and sesame oil is about 60-65 mg/mL; when the weight ratio ofbenzyl benzoate to sesame oil is about 1.1, the solubility of SDE whenadded to the mixture of benzyl benzoate and sesame oil is about 70mg/mL; when the weight ratio of benzyl benzoate to sesame oil is about1.5, the solubility of SDE when added to the mixture of benzyl benzoateand sesame oil is about 150 mg/mL; when the weight ratio of benzylbenzoate to sesame oil is about 2.3, the solubility of SDE when added tothe mixture of benzyl benzoate and sesame oil is about 200 mg/mL; whenthe weight ratio of benzyl benzoate to sesame oil is about 9, thesolubility of SDE when added to the mixture of benzyl benzoate andsesame oil is greater than 300 mg/mL.

In some embodiments, the concentration of the nalbuphine ester prodrugin the formulation is greater than the solubility of the nalbuphineester prodrug when added to the mixture of the pharmaceuticallyacceptable oil and the oil-miscible retaining solvent. For example, theprodrug may be SDE, the retaining solvent may be benzyl benzoate, theoil may be sesame oil, and the retaining solvent/oil ratio, e.g., theBB/oil ratio, may be set to about 1 or about 0.8-1.2:1, so as to give apredetermined release period or a desired duration of action. In thiscase, the concentration of SDE in the formulation may be increased fromabout 60-65 mg/mL (the solubility of SDE in the mixture of benzylbenzoate and sesame oil with the BB/oil ratio of about 1) to about70-100 mg/mL by using the following manufacturing process: a) fullydissolving SDE in benzyl benzoate, and b) mixing the resulting solutionwith sesame oil to give a homogenous solution. In this preparativemethod of the present invention, SDE is first dissolved in benzylbenzoate to form a clear solution which is then mixed with sesame oil togive a homogeneous solution, by which a homogenous and stableformulation having a BB/oil ratio of about 0.8-1.2:1 and a highconcentration of SDE (such as 70-100 mg/mL) can be prepared. Theformulations prepared accordingly are stable and have a long shelf-life,staying homogenous and free from forming solid particles or precipitateseven after being stored at 2-8° C. for at least 24 months. Therefore,the formulations are suitable for administration by intramuscularinjection.

In some embodiments, the retaining solvent/oil ratio is equal to orgreater than about 1.1:1. For example, the prodrug may be SDE, theretaining solvent may be benzyl benzoate, the oil may be sesame oil, andthe retaining solvent/oil ratio, e.g., the BB/oil ratio, may be set toabout 1.1, so as to give a predetermined release period or a desiredduration of action. In some embodiments, the concentration of thenalbuphine ester prodrug in the formulation is greater than thesolubility of the nalbuphine ester prodrug when added to the mixture ofthe pharmaceutically acceptable oil and the oil-miscible retainingsolvent and the retaining solvent/oil ratio is equal to or greater thanabout 1.1:1.

In one embodiment, the nalbuphine ester prodrug is SDE; thepharmaceutically acceptable oil is sesame oil; the oil-miscibleretaining solvent is benzyl benzoate; the weight ratio of benzylbenzoate to sesame oil is about 1.1-3:1; and the concentration of SDE inthe formulation is greater than about 70 mg/mL.

In some embodiments, the retaining solvent/oil ratio is equal to orgreater than about 1.5:1. In some embodiments, the concentration of thenalbuphine ester prodrug in the formulation is greater than thesolubility of the nalbuphine ester prodrug when added to the mixture ofthe pharmaceutically acceptable oil and the oil-miscible retainingsolvent and the retaining solvent/oil ratio is equal to or greater thanabout 1.5:1. In one embodiment, the nalbuphine ester prodrug is SDE, thepharmaceutically acceptable oil is sesame oil, and the oil-miscibleretaining solvent is benzyl benzoate.

It should be understood that when the BB/oil is increased, for example,from about 1 to about 1.5, the solubility of SDE in the formulation willbe increased significantly, for example, from about 60 mg/mL to about150 mg/mL. When the formulation is intended to be prepared with a SDEconcentration greater than the solubility of SDE when added to themixture of benzyl benzoate and sesame oil, the homogeneously dissolvedsolution can only be prepared by mixing SDE with benzyl benzoate to forma clear solution, and then mixing the clear solution with sesame oil.When the formulation is intended to be prepared with a SDE concentrationequal to or lower than the solubility of SDE when added to the mixtureof benzyl benzoate and sesame oil, the homogeneously dissolved solutionmay be given either by mixing SDE directly with the mixture of benzylbenzoate and sesame oil, or by pre-dissolving SDE with benzyl benzoate.

In some embodiments, the BB/oil ratio is equal to or greater than about1.1:1, and the concentration of SDE in the formulation is greater thanabout 70 mg/mL. For example, the BB/oil ratio may be about 1.1:1, andthe concentration of SDE in the formulation may be about 70-100 mg/mL.

In some embodiments, the nalbuphine ester prodrug is SDE; thepharmaceutically acceptable oil is sesame oil and the oil-miscibleretaining solvent is benzyl benzoate; the weight ratio of benzylbenzoate to sesame oil is about 0.8-1.2:1; and the concentration of SDEin the formulation is greater than about 70 mg/mL.

In some embodiments, the concentration of SDE in the formulation isabout 70-80 mg/mL, and the weight ratio of benzyl benzoate to sesame oilis about 1.1-1.2:1. In one embodiment, the concentration of SDE in theformulation is about 75 mg/mL, and the weight ratio of benzyl benzoateto sesame oil is about 1.12:1. In one embodiment, the concentration ofSDE in the formulation is about 80 mg/mL, and the weight ratio of benzylbenzoate to sesame oil is about 1.18:1.

In some embodiments, the formulations can further comprise asolubilizing agent. The solubilizing agent may help to increase theconcentration of the nalbuphine ester prodrug in the formulation; in themeantime, the solubilizing agent is miscible with therelease-controlling solution. For example, the solubilizing agent may bean alkyl alcohol. When the alkyl alcohol is used independently todissolve the nalbuphine ester prodrug, the solubility of the prodrug inthe alkyl alcohol may be relatively low. For example, the solubilitiesof SDE in ethanol, 1-propanol and t-butanol are about 16 mg/mL, about 32mg/mL, and about 19 mg/mL, respectively. However, in some embodiments,when the alkyl alcohol is added into the present formulations, thesolubility of the prodrug in the formulations may be increased by atleast 30%. For example, the solubility of SDE in the formulations may beincreased from about 160 mg/mL to about 210 mg/mL when 10% by weight oftert-Butanol is used to replace the corresponding amount of benzylbenzoate in the formulations.

The solubilizing agent may be a C2-C6 alkyl alcohol or any mixturethereof. The C2-C6 alkyl alcohol may be a linear or branched alkylalcohol. In some embodiments, the alkyl alcohol may be a C2-C5 alkylalcohol, such as ethanol, 1-propanol, isopropanol, 1-butanol,sec-butanol, isobutanol, tert-butanol, n-pentanol, 2-pentanol,3-pentanol, 2-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol,3-methyl-1-butanol, and/or 2,2-dimethyl-1-propanol. In some embodiments,the alkyl alcohol may be ethanol, 1-propanol, 2-propanol, 1-butanol,and/or tert-butanol. In some embodiments, the w/w % of the alkyl alcoholin the solvent system (i.e., the sum of the alkyl alcohol, the retainingsolvent and the oil) is about 2.5-30%, for example, 2.5%, 5%, 10%, 15%,20% or 30%. In some embodiments, the w/w % of the alkyl alcohol in thesolvent system is about 10-20%. In some embodiments, the solubilizingagent may be used to increase the solubility of nalbuphine ester prodrugin the final formulations to more than 100 mg/mL, for example, more than150, 200, 250, or 300 mg/mL. In some embodiments, the solubilizing agentmay be used to increase the concentration of nalbuphine ester prodrug inthe final formulations by at least 30%.

It should be understood that the preparation of an oil-containingformulation with a high concentration of SDE is challenging due to thelow solubility of SDE in the oil. Clinically, 5 mL has been cited forhuman adults as the maximum volume for a single IM injection. Whenpreparing an injectable extended release formulation of SDE, the longerthe effective release period is expected to be, the higher the drug loadshould be. In the situation that the injection volume is limited to lessthan 5 mL (even lower for adults with less muscle mass), theconcentration of SDE must be increased in order to increase the drugload. The pre-dissolving of SDE in the retaining solvent and theaddition of the solubilizing agent, particularly ethanol, may,respectively, significantly increase the solubility of SDE in theformulation, by which a long-term release formulation of SDE with a highdrug-loading can be achieved.

In some embodiments, the pharmaceutical formulations of the presentinvention each comprise SDE, the release-controlling solution, and thesolubilizing agent. In some examples, the release-controlling solutioncomprises the pharmaceutically acceptable oil selected from the groupconsisting of sesame oil, castor oil, and cottonseed oil; and theoil-miscible retaining solvent containing benzyl benzoate or benzylalcohol. In some examples, the solubilizing agent is an alkyl alcoholselected from the group consisting of ethanol, 1-propanol, 2-propanol,1-butanol, and tert-butanol. In some examples, the w/w % of thepharmaceutically acceptable oil in the solvent system (i.e., the sum ofthe oil, the retaining solvent, and the solubilizing agent) is about 5%,10%, 20%, 30%, 40%, 45%, 50%, 55%, or 60%. In some examples, the w/w %of the retaining solvent in the solvent system is about 30%, 35%, 37.5%,40%, 45%, 47.5%, 50%, 55%, 57.5%, 60%, 65%, 67.5%, 70%, 75%, 77.5%, 80%,or 85%. In some examples, the w/w % of the solubilizing agent in thesolvent system is about 2.5%, 5%, 10%, 15%, 20%, or 30%. In someexamples, the concentration of SDE in the formulation is above 100mg/mL, and the solvent system comprises about 2.5-30 w/w % of the alkylalcohol. In some examples, the concentration of SDE in the formulationis above 150 mg/mL, and the solvent system comprises about 5-30% of thealkyl alcohol. In some examples, the concentration of SDE in theformulation is above 200 mg/mL, and the solvent system comprises about10-20% of the alkyl alcohol. The formulation may be administered intoanimals or humans by subcutaneous or intramuscular injection.

The addition of the alkyl alcohol enables the present formulations to beprepared at a higher SDE concentration while showing excellentstability. In some embodiments, the present pharmaceutical formulationsare free from forming solid particles or precipitates after being storedat 2-8° C. for at least 24 hours. Moreover, the addition of the alcoholmay help to decrease the viscosity of the present formulations, so as toallow an easier injection through a small gauge needle.

In some embodiments, the formulations may further comprise aneutralizing agent, with or without a solubilizing agent. Theneutralizing agent may help in neutralizing the acid generated duringthe conversion of nalbuphine ester prodrug to nalbuphine. For example,when SDE is converted into nalbuphine, sebacoyl acid will be generated.Accumulation of sebacoyl acid may cause injection site irritation. Theaddition of a neutralizing agent may help to neutralize sebacoyl acid soas to ease the local irritation at the injection site. The neutralizingagent may be a pharmaceutically acceptable basic salt. For example, thebasic salt may be a citrate salt, phosphate salt, carbonate salt,lactate salt, tartrate salt, or succinate salt. In some embodiments, thebasic salt may be a sodium salt or a potassium salt. In someembodiments, the basic salt may be trisodium citrate, disodiumphosphate, sodium bicarbonate, or sodium lactate.

In some embodiments, the pharmaceutical formulations of the presentinvention each comprise SDE, the release-controlling solution, thesolubilizing agent, and the neutralizing agent.

The present pharmaceutical formulations may further comprise suitableinactive ingredients, pharmaceutically or veterinary acceptablecarriers, including but not limited to viscosity modifiers, coloring,and flavoring agents, etc.

Methods of Preparation

In some embodiments, the pharmaceutical formulations of the presentinvention may be prepared without heating steps or complicated mixingsequences.

The present invention also provides methods of preparing an extendedrelease formulation of nalbuphine ester prodrug with a predeterminedrelease period, each comprising:

-   -   1) providing an oil-miscible retaining solvent and a        pharmaceutically acceptable oil, wherein the weight ratio of the        oil-miscible retaining solvent to the pharmaceutically        acceptable oil (the “retaining solvent/oil ratio”) is adjusted        based on the predetermined release period; and    -   2) mixing the nalbuphine ester prodrug with the oil-miscible        retaining solvent and the pharmaceutically acceptable oil, to        form a homogeneously dissolved solution.

In some embodiments, the step of mixing the prodrug with the retainingsolvent and the oil comprises mixing the prodrug with a mixture of theoil-miscible retaining solvent and the pharmaceutically acceptable oil.For example, when the predetermined release period is set to greaterthan 14 days, the retaining solvent/oil ratio may be adjusted to greaterthan 1. For example, the retaining solvent/oil ratio may be about 2. Inone embodiment, a release-controlling solution, i.e. the mixture of theretaining solvent and the oil, comprising about 12 g of the retainingsolvent and about 6 g of the oil may be firstly prepared and then mixedwith about 1.6 g of the prodrug.

In some embodiments, the step of mixing the prodrug with the retainingsolvent and the oil comprises the steps of (a) dissolving the nalbuphineester prodrug in the oil-miscible retaining solvent thereby giving analbuphine ester prodrug solution (a clear solution), and (b) mixing thetherapeutically acceptable oil with the nalbuphine ester prodrugsolution resulting from step (a) to give a homogenously dissolvedsolution. In some embodiments, when the predetermined release period isset to lower than 14 days, the retaining solvent/oil ratio may beadjusted to lower than 1. For example, the retaining solvent/oil ratiomay be about 0.65. In one embodiment, about 1.5 g of the prodrug may bedissolved in about 11 g of the retaining solvent to give the clearsolution, and the clear solution is then mixed with about 17 g of theoil. In some embodiments, the concentration of the prodrug in theformulation is higher than the solubility of the prodrug when added tothe mixture of the retaining solvent and the oil. For example, when theBB/oil ratio is about 1.1, the SDE concentration in the formulation maybe about 75 mg/mL or about 80 mg/mL (the solubility of SDE when addedinto the mixture of benzyl benzoate and sesame oil with a BB/oil ratioof about 1.1 is about 70 mg/mL).

As described above, the nalbuphine ester prodrug is more soluble informulations with increased retaining solvent/oil ratios, and theduration of drug release is longer for such formulations. When a longerrelease period is desired, the retaining solvent/oil ratio may beadjusted to a higher value, where the solubility of the prodrug whenadded to the mixture of the retaining solvent and the oil may be higherthan the concentration of the prodrug in the final formulation;accordingly, such a formulation may be prepared either by directlymixing the prodrug with the release-controlling solution (i.e. themixture of the retaining solvent and the oil), or by pre-dissolving theprodrug in the retaining solvent. However, when a shorter release periodin expected, i.e., the retaining solvent/oil ratio is lower, or theintended concentration of the prodrug in the final formulation is equalto or greater than the saturated concentration (i.e. the solubility ofthe prodrug when added to the mixture of the retaining solvent and theoil), the formulation can be prepared by pre-dissolving the prodrug inthe retaining solvent, so that the solubility of the prodrug in theformulation may be significantly improved. As such, the concentration ofthe prodrug in the resulting formulation may be greater than thesolubility of the prodrug when added to a solution of the retainingsolvent and oil.

Once the homogeneously dissolved solutions are formed by the preparativemethods of the present invention, i.e., either by directly mixing theprodrug with the release-controlling solution or by pre-dissolving theprodrug in the retaining solvent, they may all be stable solutions. Insome embodiments, the formulations prepared by pre-dissolving SDE inbenzyl benzoate and then mixing with sesame oil are stable at 2-8° C.for at 24 months, and may remain homogeneous at about 0-4° C. while notforming precipitates or solid particles.

In some embodiments of the methods, the nalbuphine ester prodrug is SDE.

In some embodiments, step (a) may further include stirring the mixtureof SDE and the oil-miscible retaining solvent for about 30 to 90minutes, for example, for about 60 minutes. In some embodiments, step(b) may further include stirring the mixture of the resulting solutionof step (a) and the oil for about 15 to 45 minutes, for example, forabout 30 minutes. In some embodiments, step (a) and step (b) may beconducted at room temperature. The formulations of the present inventionmay be prepared without any heating process or other commonly usedtechniques to cause SDE to fully dissolve, by which a high purity of theresulting formulation can be achieved. Moreover, in some embodiments,only a short period of stirring is needed to prepare formulations of thepresent invention. Accordingly, the formulations of the presentinvention may be prepared in a more economical and convenient manner,which is beneficial to large-scale production.

In some embodiments of the methods, the oil-miscible retaining solventmay be benzyl benzoate, benzyl alcohol, or any mixture thereof, and thepharmaceutically acceptable oil may be sesame oil, cottonseed oil,castor oil, or any mixture thereof.

The present invention also provides a method for preparing apharmaceutical formulation, comprising dissolving the nalbuphine esterprodrug in the oil-miscible retaining solvent; and mixing the resultingsolution with the pharmaceutically acceptable oil to give a homogenoussolution, wherein the formulation is suitable for administration byinjection. In some embodiments, the nalbuphine ester prodrug is SDE; theconcentration of SDE in the formulation is greater than about 70 mg/mL;the weight ratio of benzyl benzoate to sesame oil is about 0.8-1.2:1;and the formulation is suitable for administration by intramuscular orsubcutaneous injection.

In some embodiments, the retaining solvent may be benzyl benzoate andthe oil may be sesame oil, where the weight ratio of benzyl benzoate tothe oil (the “BB/oil ratio”) may be about 0.5 to about 16. In someembodiments, the BB/oil ratio is about 0.65 to about 3. In someembodiments, the BB/oil ratio is about 0.65 to about 2. In otherembodiments, the BB/oil ratio is about 1-3 or about 1-2. In someembodiments, when the release period is predetermined to give a durationof action of about 5 or 6 days, the BB/oil ratio may be adjusted toabout 1.1. Accordingly, when the release period is predetermined to givea duration of action of more than 6 days, the BB/oil ratio may beadjusted to more than about 1.1; and when the release period ispredetermined to give a duration of action of less than 6 days, theBB/oil ratio may be adjusted to less than about 1.1.

The methods may each further comprise adding a solubilizing agent and/ora neutralizing agent. In some embodiments, the solubilizing agent and/orthe neutralizing agent may be added to the release-controlling solutionbefore the mixing with the prodrug. For example, the solubilizing agentand/or the neutralizing agent may be added to the release-controllingsolution, and then mixed with the prodrug. Alternatively, theformulations may be prepared by mixing the prodrug with the mixture ofthe retaining solvent and the solubilizing agent and/or the neutralizingagent first, and then mixing with the oil.

The pharmaceutically acceptable oils, the oil-miscible retainingsolvents, the solubilizing agents, and the neutralizing agents used inthe methods of the present invention are the same as those described forthe present formulations.

In some embodiments of the methods, the solubilizing agent may be analkyl alcohol, such as ethanol, 1-propanol, 2-propanol, 1-butanol,and/or tert-butanol; and the nalbuphine ester prodrug may be SDE. Insome embodiments, the solubilizing agent may be ethanol. The addition ofthe alkyl alcohol may significantly increase the solubility of SDE inthe final formulations, by which a high drug loading formulation with along release period can be achieved. In some embodiments, the alkylalcohol may be used to increase the concentration of SDE in the finalformulations to more than 100 mg/mL, for example, more than 150, 200,250, or 300 mg/mL. In some embodiments, the alkyl alcohol may be used toincrease the concentration of SDE in the final formulations by at least30%. The alkyl alcohol may be added into the release-controllingsolution before SDE is added into the solvent system. Alternatively, thealkyl alcohol may be added into the retaining solvent before SDE ismixed with the retaining solvent.

In some embodiments of the methods, the neutralizing agent may be abasic salt selected from the group consisting of trisodium citrate,disodium phosphate, sodium bicarbonate, and sodium lactate. The basicsalt may be added into the formulation before or after mixing theprodrug with the release-controlling solution. Alternatively, the basicsalt may be added into the formulation before or after the retainingsolvent is mixed with the prodrug. Alternatively, the neutralizing agentmay be mixed with the formulation prior to the administration of thepresent formulation.

In some embodiments, the methods of the present invention may furthercomprise filtering the homogenously dissolved solution with a bacteriaproof filter, such as a Millipore 0.22 μm filter. The formulations ofthe present invention are homogeneously dissolved solutions withoutprecipitates or solid particles, so that they can be easily sterilizedby filtration with full or nearly full recovery of the drug.

Extended Release Periods/Lower Release Rates

In some embodiments, the present formulations provide extended releaseperiods of nalbuphine ester prodrugs. For example, release rates/periodsof nalbuphine ester prodrugs may be demonstrated or estimated through invitro dissolution experiments designed to cause nalbuphine ester prodrugrelease from the formulations at a higher rate than the actual releaserate in a living subject. When a formulation shows a lower dissolutionrate in the dissolution experiments, it is expected that the formulationmay have a longer release period (or a lower release rate) in a livingsubject. The dissolution rate may be defined as the amount of theprodrug, e.g., SDE, which goes into a dissolution medium from theformulation per unit time under certain conditions of interface,temperature and solvent composition. The dissolution of the nalbuphineester prodrug can be determined by carefully dropping the formulationinto a larger volume of a dissolution medium. For example, the volume offormulation may be 50-150 μl and the volume of dissolution medium may be200-1000 ml, for example 500 ml. The dissolution medium may be a bufferwith a surfactant, for example phosphate-buffered saline with 1% tween80 and a pH of 6.0 (PBST). The dissolution medium can then be stirredand samples of the resulting medium can be withdrawn at predeterminedtime intervals. Prior to termination of the experiment, HCl can be addedto the resulting medium to allow 100% release of the nalphubine esterprodrug and a sample can be taken and used as a reference point of 100%in calculating the release rate of the nalbuphine ester prodrug. Thenalbuphine ester prodrug can be SDE. For example, the dissolutionprofile of a formulation can be determined as described in Example 1.

In some embodiments, when the retaining solvent/oil ratio is about 0.5,about 0.65, about 1, about 2, about 3, and about 16, the time forreleasing about 50% of the total amount of the nalbuphine ester prodrugfrom the formulation into the in vitro medium may be about 15-25minutes, about 20-30 minutes, about 35-45 minutes, about 50-60 minutes,about 85-95 minutes, and about 120-130 minutes, respectively. It isdemonstrated that when the retaining solvent/oil ratio is increased, thedissolution rate of the prodrug from the formulation may be decreased.The lower in vitro dissolution rate of a formulation may reflect alonger release period in a living subject (or a lower in vivo releaserate).

The release rates/periods of the present formulations, or the aforesaidcorrelation between the in vitro dissolution rate and the in vivorelease period/release rate, may be further assessed or verified byevaluating the in vivo availability and pharmacokinetic parameters ofnalbuphine after administration of a present formulation to a livingsubject. For example, a nalbuphine ester prodrug formulation of thepresent invention can be administered to a living subject through aninjection. The nalbuphine ester prodrug can be SDE. In some embodiments,the injection may be subcutaneous. In other embodiments, the injectionmay be intramuscular. The subject may be an animal, for example a dog,cat, or rodent, or the subject may be a human. Blood samples may betaken from the subjects prior to formulation administration and atvarious time points following administration of the formulation, such asover the first 144 or 360 hours after administration. For example, bloodsamples may be drawn at 1, 2, 6, 24, 36, 48, 60, 72, 96, 120 and 144hours after administration. Alternatively, blood samples may be drawn at0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours afteradministration or at similar intervals after administration, and atregular intervals for up to 12 days after administration. The wholeblood or plasma concentration of nalbuphine can then be determined forthe blood samples. For example, plasma and whole blood concentrations ofnalbuphine can be determined as described in Examples 3 and 4.

In some embodiments, when the retaining solvent/oil ratio is adjusted toabout 0.65, about 99% of the prodrug may be released from theformulation at 144 hours from dosing; when the retaining solvent/oilratio is adjusted to about 1, about 90% of the prodrug may be releasedfrom the formulation at 144 hours from dosing; when the retainingsolvent/oil ratio is adjusted to about 2, about 80% of the prodrug maybe released from the formulation at 144 hours from dosing. It is shownthat when the retaining solvent/oil ratio is increased, the in vivorelease period of the formulation may be extended or prolonged, which isconsistent with the results demonstrated by the in vitro dissolutionexperiments.

In some embodiments, when the retaining solvent/oil ratio is adjusted toabout 1, e.g., 1.12, the present formulation may have a release periodof equal to or greater than about 10 days, 12 days and 14 days and aduration of action of equal to or greater than about 5 or 6 days. Insome embodiments, the retaining solvent/oil ratio may be adjusted tolower than about 1, e.g., 0.65, to give a formulation having a durationof action of less than 6 days, for example, about 4 days. In otherembodiments, the retaining solvent/oil ratio may be adjusted to morethan about 1.1, e.g., 2, to give a formulation having a duration ofaction of equal to or greater than about 5 days and 6 days, for example,more than 6 days.

In one embodiment, a pharmaceutical formulation of the present inventionis administered intramuscularly into a subject in need thereof for painrelief and the plasma or whole blood concentration of nalbuphine mayreach no less than 1 ng/mL within 6-12 hours of dosing and thisconcentration may remain for equal to or greater than 12 days fromadministration. In one embodiment, the plasma or whole bloodconcentration of nalbuphine may reach no less than 3 ng/mL within 12-24hours of dosing and this concentration of nalbuphine may be maintainedfor equal to or greater than 7 days from dosing. In another embodiment,the plasma or whole blood concentration of nalbuphine may reach no lessthan 3 ng/mL within 12-24 hours of dosing, and maintain at saidconcentration for equal to or greater than 9 days from dosing.

Pharmacokinetic parameters may also be calculated from the whole bloodor plasma nalbuphine concentrations, for example, the maximum observednalbuphine concentration in the plasma or whole blood (C_(max)) may bedetermined. The time to C_(max) (T_(max)), half-life (T_(1/2)), meanresidence time (MRT), and the area under the curve (time zero to thelast quantifiable nalbuphine measurement (AUC_(0-t)) and time zeroextrapolated to infinity (AUC_(0-inf))) may also be determined. Forexample, plasma and whole blood concentrations of nalbuphine and thepharmacokinetic parameters of the present formulations can be determinedas described in Examples 3 and 4.

After administration via intramuscular injection to a subject, in someembodiments the formulations of the present invention exhibit thefollowing in vivo characteristics: (a) a peak plasma level of nalbuphineoccurs within 45-66 hours after administration (T_(max)); and (b) anaverage elimination half-life (T_(1/2)) of nalbuphine afteradministration is about 56-90 hours.

In some embodiments, the plasma or whole blood concentrations ofnalbuphine following administration may be used to determine the onsetof the analgesic effect and the duration of action of the presentformulations. For example, the obtained plasma or whole bloodconcentrations of nalbuphine may be compared to a concentration ofplasma or whole blood nalbuphine that has been reported to exhibitanalgesic effects. In general, by administration of a pharmaceuticalformulation of this disclosure via intramuscular injection, the onset ofthe analgesic effect may take place in about 6 to 36 hours of dosing andthe duration of action may be about 6 to about 12 days. In someembodiments, the duration of action may be equal to or greater thanabout 5 days, about 6 days, about 9 days, or about 12 days. In someembodiments, the duration of action may be equal to or greater than 3,6, 9, 10, 12, or 14 days. In some embodiments, the duration of action ofthe formulation is equal to or greater than about 5 or 6 days. In otherembodiments, the duration of action of the formulation is equal to orgreater than about 7 days. The time of onset for an analgesic effect candepend on dosing, the individual's response, and the type of pain reliefsought.

In certain embodiments, the present formulation is prepared with aBB/oil ratio of about 0.8-1.2, wherein the SDE concentration is about 75mg/mL. In some embodiments, such formulations may maintain a bloodconcentration of nalbuphine at >3 ng/mL for equal to or greater thanabout 6 days, or at >1 ng/mL for equal to or greater than about 12 days,when a single dose of 150 mg of SDE is administered. Formulations withan increased BB/oil ratio may have a duration of action for more than 6days. Similarly, if the formulation is intended to provide a duration ofaction shorter than 6 days, e.g., 3 or 5 days, the BB/oil ratio may bedecreased.

It should be understood that the duration of action of the presentformulation may depend not only on the retaining solvent/oil ratio, butalso the concentration of the prodrug in the formulation, and the totaldose being administered. The concentration of the prodrug in theformulation, and the total dose being administered may be adjusted perneed, so as to provide an effective blood concentration for a desiredperiod.

Treatment of Pain

In another aspect, the present invention provides methods for treatingpain, comprising administering a therapeutically effective amount of thepharmaceutical formulation of the present invention to a subject in needthereof. In some embodiments, the present invention provides methods fortreating post-surgical pain or other types of long-term pain by thepresent formulations. In some embodiments, the pharmaceuticalformulation is administered by intramuscular or subcutaneous injection.Treatment of pain can be evaluated by assessing a subject's assessmentof pain on an intensity scale. For example, the Visual Analog Scale(VAS) for pain can be used to score a subject's pain intensity (PsycholMed. 1988 November; 18(4):1007-19). The pain VAS is a unidimensionalintensity scale on which the subject is asked to indicate his or herlevel of pain intensity. For example, pain intensity may be assessedstarted right before the first use of patient-controlled analgesia(PCA), and at 1±0.1, 2±0.1, 3±0.1, 4±0.25, 8±0.5, 12±0.5, 16±0.5,20±0.5, 24±1, 28±1, 32±2, 36±2, 40±2, 44±2, 48±2 hours after thesurgery, and be assessed during Days 3-7 in the morning and evening, aswell as during special events such as bowel movements. For example, thepatient-controlled analgesic is ketorolac. In some embodiments, thepresent formulations may be administered to a subject prior to asurgical procedure. For example, the surgical procedure may be ahemorrhoidectomy. In certain embodiments, the present formulations maybe administered 6-36 hours prior to surgery. In some embodiments, 150 mgof SDE is administered prior to surgery. Pain intensity can then beassessed following surgery, for example for 48 hours after surgery. Insome embodiments, pain can be assessed with the VAS pain scale. In someembodiments, subjects administered a present formulation have a lowerVAS pain score over 48 hours than subjects administered a placebo. Forexample, evaluation of the treatment of pain can be determined asdescribed in Example 4.

Treatment of pain can also be assessed by evaluating a subject's use ofa post-surgical analgesic, i.e. a rescue medicine, such as ketorolac.For example, the time of a subject's first use of the post-surgicalanalgesic following surgery can be evaluated. In some embodiments,subjects administered a present formulation prior to surgery have alonger period of time before the post-surgical analgesic use compared tosubjects administered a placebo. For example, treatment of pain can beassessed as described in Example 4.

Treatment of pain can also be assessed by evaluating a subject'seffective blood concentration of nalbuphine after administration of thepresent formulation. In some embodiments, when the retaining solvent/oilratio is increased, the time to reach the effective blood concentrationof nalbuphine may be longer, thus the onset of action of the formulationmay be slower. Accordingly, the time to administer the formulation intoa subject in need may be adjusted per the desired onset of action of theformulation. For example, when the retaining solvent/oil ratio is about0.65, about 1, or about 2, the blood concentration of nalbuphine at 1hour after administration may be about 28 ng/mL, about 11 ng/mL, orabout 7 ng/mL, respectively. In some embodiments, for example, if theeffective blood concentration of nalbuphine in a living subject, e.g., adog, is about 5 ng/mL, the formulation having a retaining solvent/oilratio of about 2 may be administered to the subject about 1 hour priorto the onset of pain symptom; while the formulation having a retainingsolvent/oil ratio of about 0.65 may be administered to the subject about30 minutes prior to the onset of pain symptom.

In some embodiments, the present methods for treating pain comprisesadministering to a subject, e.g., a human, in need thereof theformulation of the present invention having a retaining solvent/oilratio of about 0.8-1.2, e.g., about 1.12, at 6 to 36 hours prior to theonset of pain symptoms, wherein the formulation is administered to thesubject via intramuscular injection. In some embodiments, theformulation having a retaining solvent/oil ratio of about 1.12 may beadministered 12 to 36 hours prior to the onset of pain symptoms. In someother embodiments, the formulation having a retaining solvent/oil ratioof about 1.12 may be administered 12 to 24 hours prior to the onset ofpain symptoms. In some embodiments, the concentration of SDE in theformulation is about 75 mg/mL, and such formulation is administered witha total dose of up to 150 mg of SDE.

In some embodiments, the present methods for treating pain comprisesadministering to a subject, e.g., a dog, in need thereof the formulationof the present invention having a retaining solvent/oil ratio of about1.18. In one embodiment, the concentration of SDE in the formulation isabout 80 mg/mL, and such formulation is administered with a total doseof up to 160 mg of SDE.

In some embodiments, the present method is for treating post-surgicalpain, wherein the onset of pain symptom is during or after a surgicaloperation on the subject. Examples of the surgical operation includecommon types of general surgery, such as hernia surgery, hemorrhoidsurgery, abdominal surgery, obstetric and gynecological surgery, plasticsurgery, orthopedic surgery, otolaryngology surgery, male genitalprocedures, and dental surgery. In some embodiments, the surgicaloperation is hemorrhoid surgery. In certain embodiments, the formulationhaving a retaining solvent/oil ratio of about 1.12 is administered 12 to36 hours prior to the surgical operation. In some embodiments, theformulation having a retaining solvent/oil ratio of about 1.12 isadministered 12 to 24 hours prior to the surgical operation. In someembodiments, the formulation being administered contains SDE, sesame oiland benzyl benzoate, where the weight ratio of benzyl benzoate to sesameoil is about 0.8-1.2:1; and the concentration of SDE in the formulationis about 75 mg/mL. In certain embodiments, a formulation having aretaining solvent/oil ratio of about 1.12 is administered to a humanwith a total dose of up to 150 mg of SDE.

Another aspect of the present invention is to provide a method oftreating long-term pain, which includes administering to a subject inneed thereof via intramuscular injection a pharmaceutical formulation ofthe present invention having a retaining solvent/oil ratio of about 1.12at 6-36 hours prior to the onset of pain symptoms. Examples of thelong-term pain include labor pain, chronic back pain, and chronic jointpain. In some embodiments, the formulation having a retainingsolvent/oil ratio of about 1.12 is administered about 12-36 hours priorto the onset of pain symptoms. In certain embodiments, the formulationhaving a retaining solvent/oil ratio of about 1.12 is administered witha total dose of up to 150 mg of SDE.

In some embodiments, the formulation is prepared with a retainingsolvent/oil ratio of about 0.8-1.2 and a SDE concentration of about 75mg/mL, and is intramuscularly administered with a total dose of up toabout 150 mg of SDE. In such case, the onset of action of theformulation may be within about 6-36 hours, or about 12-36 hours, fromdosing; and the duration of action of the formulation may be about 6days, about 7 days, or about 9 days. In other embodiments, theformulation is prepared with a retaining solvent/oil ratio of about 0.65and a SDE concentration of about 50 mg/mL, and is intramuscularlyadministered with a total dose of up to about 160 mg of SDE. In otherembodiments, the formulation is prepared with a retaining solvent/oilratio of about 2 and a SDE concentration of about 80 mg/mL, and isintramuscularly administered with a total dose of up to about 160 mg ofSDE.

In some embodiments, the formulation is administered at a concentrationof about 50-160 mg/ml, or about 70-100 mg/mL, with a total administereddose of about 75-160 mg, or about 150-160 mg. For example, theconcentration may be about 50, 75, 80, 100, or 150 mg/mL; and the totaldose may be about 75, 100, 125, 150, or 160 mg. In certain embodiments,the formulation is administered at a concentration of about 75 mg/ml anda total dose of about 150 mg. In other embodiments, the formulation isadministered at a concentration of about 50 mg/mL, or about 80 mg/ml,and a total dose of about 160 mg.

EXAMPLES Example 1. In Vitro Release Study of Present Formulations withVarious BB/Oil Ratio

(1) Preparation of the Present Formulations

Eight present formulations with various SDE concentrations (50-150mg/mL) and various weight ratios of benzyl benzoate to sesame oil(“BB/oil ratio”), or the mixture of benzyl benzoate and benzyl alcoholto sesame oil (“BB+BA/oil ratio”) (0.5-19) were prepared based on Table1A and Table 1B. Among them, AF4, R2, N7 and N9 formulations wereprepared according to the following Method A.

Method A:

The solvent systems (i.e., the mixture of benzyl benzoate, with orwithout benzyl alcohol, and sesame oil) were respectively prepared bymixing each component with the predetermined volume corresponding to thedesired weight/weight percent (w/w %) listed in Table 1A and Table 1B.The resulting solvent mixtures were vortexed or stirred at roomtemperature to fully mix each component. The predetermined amounts ofSDE were respectively weighed, based on the SDE concentrations listed inTable 1A and 1B, and then added into volumetric flasks containing thecorresponding solvent mixtures. The resulting mixtures were fully mixedby inverting and shaking the volumetric flasks to give the finalformulations.

R1, AF1, AF3 and N8 formulations were prepared according to thefollowing Method B.

Method B:

The predetermined amounts of SDE were respectively weighed, based on theSDE concentrations listed in Table 1A and Table 1B, and then added intobenzyl benzoate, or the mixture of benzyl benzoate and benzyl alcohol,of predetermined volume corresponding to the desired weight/weightpercent (w/w %) listed in Table 1A and Table 1B. The resulting mixtureswere mixed by vortexing or stirring to fully dissolve SDE. Sesame oil ofpredetermined volume was then added into the resulting mixtures ofbenzyl benzoate, or the mixture of benzyl benzoate and benzyl alcohol,and SDE to give the final formulations.

TABLE 1A The present formulations having various BB/oil ratios SampleBenzyl benzoate Sesame oil SDE BB/oil No. (w/w %) (w/w %) (mg/mL) ratioR1 33 67 50 0.5 AF3 39 61 50 0.65 AF1 54.2 45.8 80 1.18 AF4 67 33 80 2R2 75 25 75 3 N7 94 6 150 16

TABLE 1B The present formulations having various BB + BA/oil ratiosBenzyl benzoate + Benzyl alcohol Sesame oil SDE BB + BA/oil (w/w %) (w/w%) (mg/mL) ratio N8 45 55 75 0.82 N9 95 5 150 19

(2) In Vitro Dissolution Experiment:

The following in vitro dissolution experiment and UPLC analysis wereperformed on the formulations listed in Table 1A and Table 1B.

The dissolution/release rate of SDE from each of the formulations wasrespectively assessed by in vitro dissolution experiment. Thedissolution medium was a PBST buffer (pH6.0) with 0.1% tween 80. Foreach experiment, 500 mL medium was placed in a 600 mL beaker and the airbubble on the top was removed. 50-150 μl of each SDE formulation listedin Table 1A and Table 1B was respectively and carefully dropped into themedium (in each experiment, the dropped formulation contained the sameamount of SDE, i.e. about 7.5 mg). The temperature of the medium was setat room temperature (about 25-28° C.), and the stir speed was set atabout 360 rpm. 5 mL of the resulting medium was withdrawn atpredetermined time intervals. 200 μl 6N HCl was added into the resultingmedium 20 minutes before the termination point of the experiment toallow 100% release of SDE at an acidic condition. 5 mL of the resultingmedium at the termination point was collected and taken as the referencepoint of 100% release in calculating the dissolution rate of SDE at eachof the time intervals.

UPLC Analysis

The concentration of SDE in each of the samples collected in the invitro dissolution experiment was determined by ultra performance liquidchromatography (UPLC). Standard solutions were prepared. UPLC analysiswas conducted by using the ACQUITY UPLC ethylene bridged hybrid (BEH)C18, 1.7 μm, 2.1*50 mm column under the following conditions:

Flow rate 0.1 mL/min Injection volume 10 μl Run time 15 minutes DetectorUV 280 nm Column temperature 35° C. Sample temperature 15° C. Mobilephase Buffer A*/methanol = 40/60 *Buffer A: acetate buffer.

The accumulated SDE dissolution profiles of each of the formulations areplotted in FIG. 1A and FIG. 1B.

In FIG. 1A, it can be seen that when the BB/oil ratio is increased from0.5 to 16, the dissolution rate of SDE from the formulation isdecreased, i.e. the release period of SDE from the formulation isextended. For example, when the BB/oil ratio was about 0.5 (R1), thetime required for releasing about 50% of the total amount of SDE fromthe formulation into the in vitro medium was about 15-25 minutes; whenthe BB/oil ratio was about 3 (R2), the time required for releasing 50%of the total amount of SDE from the formulation into the in vitro mediumwas about 85-95 minutes; and when the BB/oil ratio was about 16 (N7),the time required for releasing 50% of the total amount of SDE from theformulation into the in vitro medium was about 120-130 minutes. In FIG.1B, it can be seen that the trend observed in FIG. 1A still exists whenthe retaining solvent is the mixture of benzyl benzoate and benzylalcohol.

The results indicate that the in vitro release rate/release period ofSDE from the present formulations may be regulated or controlled by theretaining solvent/oil ratio, e.g., the BB/oil ratio, of the solventsystem. When a longer release period is intended, the retainingsolvent/oil ratio in the formulation can be increased per need.

Correlations between in vitro and in vivo data are often used duringpharmaceutical development in order to reduce development time andoptimize the formulation. Many studies reported in the late '70s andearly '80s established the basic reliability of such correlations(Pharm. Res. 1990, 7, 975-982). Various definitions of in vitro-in vivocorrelation have been proposed as a predictive mathematical modeldescribing the relationship between an in vitro property of an extendedrelease dosage form (usually the rate or extent of drug dissolution orrelease) and a relevant in vivo response, e.g., plasma drugconcentration or amount of drug absorbed. Under this concept, it isbelieved that when the retaining solvent/oil ratio of the presentformulations is increased, the formulation may have anincreased/prolonged in vivo release period.

Example 2 Comparison Between Method a and Method B

(1) Suspensions with Precipitates, Prepared by Method a when the BB/OilRatio is about 1

Two samples (1 and 2) were prepared by directly adding SDE into amixture of sesame oil and benzyl benzoate (i.e., Method A of Example 1).In Sample 1, 5.5 mL sesame oil and 4.5 mL benzyl benzoate were firstmixed to form an oily solvent mixture. 750 mg of SDE was then added intothe solvent mixture. The resultant mixture was sonicated for at least 2hours, left overnight at room temperature, and then centrifuged for 10minutes at 3000 rpm. The upper clear solution was collected and thensubjected to high performance liquid chromatography (HPLC) analysis.Sample 2 was prepared by following the process for preparing Sample 1except that the amount of SDE was changed to 1000 mg. For Samples 1 and2, the ratios of “the originally added weight of SDE” to “the volume ofbenzyl benzoate and sesame oil” were 75 mg/mL and 100 mg/mL,respectively. For both of Samples 1 and 2, suspensions with precipitateswere formed immediately after SDE was added into the oily mixture. Afterbeing sonicated for over 2 hours and left overnight at room temperature,visible solid particles of SDE were still present in the resultingmixtures.

(2) Homogeneous Solutions, Prepared by Method B when the BB/Oil Ratio isabout 1

Four samples (3-6) were prepared according to Method B of Example 1.Specifically, in Sample 3, 2.25 mL of benzyl benzoate and 375 mg of SDEwere first mixed and stirred to form a clear solution. 2.75 mL of sesameoil was then added into the clear solution to give a homogeneoussolution (resulting SDE concentration of 75 mg/mL; volume ratio ofbenzyl benzoate to sesame oil being 45:55), such that the SDE ishomogenously dissolved in the solution. Sample 4 was prepared byfollowing the method for making Sample 3 except that 2 mL benzylbenzoate and 3 mL sesame oil were used (resulting SDE concentration of75 mg/mL; volume ratio of benzyl benzoate to sesame oil being 40:60).Sample 5 was prepared by following the method for making Sample 3 exceptthat 2.5 mL benzyl benzoate and 2.5 mL sesame oil were used (resultingSDE concentration of 75 mg/mL; volume ratio of benzyl benzoate to sesameoil being 50:50). Sample 6 was prepared by following the process ofmaking Sample 3 except that 500 mg SDE was used (resulting SDEconcentration of 100 mg/mL; volume ratio of benzyl benzoate to sesameoil being 45:55). Samples 3-6 were then subject to a freeze-thaw test tocheck their physical stabilities. The freeze-thaw test was conducted bycooling each of the samples at about 0-4° C. for about 12 hours, warmingeach of the cooled samples at room temperature for about 12 hours, andsequentially repeating the cooling and warming steps twice. All ofSamples 3-6 stayed clear and homogeneous after the freeze-thaw test. Theresulting samples were centrifuged for 10 minutes at 3000 rpm. The uppersolution of each sample was respectively collected and then subjected toHPLC analysis.

(3) HPLC Analysis of Samples 1-6

The HPLC analysis was conducted using the column Waters Xbridge RP18,4.6 mm×250 mm, 3.5 μm column (Part No.: 186003964) and the followingconditions:

Flow rate: 0.6 ml/min

Injection volume: 10 μl

Run Time: 70 minutes

Detector: UV wavelength 280 nm

Column temperature: 35° C.

Sample temperature: 25° C.

Gradient program:

Time (mins.) Mobile phase A % Mobile phase B % 0 50 50 30 30 70 60 30 7062 50 50 70 50 50

Mobile phase A was an acetate buffer, and Mobile phase B was methanol.1.0 mL of each of the upper solutions from centrifuged Samples 1-6 wascollected and then diluted with acetonitrile to 100 mL. The resultingsolutions were subjected to the HPLC analysis, individually andseparately. The results of the HPLC analysis were used to calculate theconcentration of dissolved SDE in centrifuged Samples 1-6, and the datais summarized in Table 2.

TABLE 2 Comparison between the formulations prepared by Method A andMethod B. Added SDE Calculated SDE Sample BB/oil concentrationConcentration # ratio Appearance (mg/mL) (mg/mL) Samples preparedaccording to Method A: 1 1.0:1 Suspension 75 60 2 1.0:1 Suspension 10065 Samples prepared according to Method B: 3 1.0:1 Clear solution 75 734 0.8:1 Clear solution 75 76 5 1.2:1 Clear solution 75 78 6 1.0:1 Clearsolution 100 102

In Table 2, the weight ratio of benzyl benzoate to sesame oil iscalculated by the following formula:

Weight ratio=Volume ratio*1.118/0.917

In the formula, 1.118 (g/cm³) represents the density of benzyl benzoate,and 0.917 (g/cm³) represents the density of sesame oil. “Added SDEconcentration” represents the ratio of “the originally added weight ofSDE” to “the originally added volume of benzyl benzoate and sesame oil.”“Calculated SDE Concentration” represents the calculated SDEconcentration of Samples 1-6, obtained by centrifuging each of thesamples, and then analyzing the resulting supernatants by HPLC.

It can be seen from Table 2 that, when the BB/oil ratio is set to about1, samples prepared according to Method A—i.e., Samples 1 and 2—were allin the form of suspension with precipitates, while samples preparedaccording to Method B—i.e., Samples 3-6—were all in a homogeneoussolution form. For Samples 1 and 2, the difference between “Added SDEconcentration” and “Calculated SDE Concentration” indicates that morethan 20% of SDE formed solid particles in the formulations, which hadgreatly exceeded the limitation of “at most 0.5-5.0% solids” acceptablefor an intramuscular injection suspension (see R. M. Patel, ParenteralSuspension: an Overview, Int. J. Curr. Pharm. Res., 2010, 2, 3:3-13). Inaddition, the visible solid particles indicated that the averageparticle size of Samples 1 and 2 was much larger than 5 micrometer(i.e., the upper limit for a qualified intramuscular injectionsuspension). Moreover, “Calculated SDE Concentration” of Samples 1 and 2indicated that the saturated concentration of SDE in the formulationsprepared by Method A, when the BB/oil ratio is set to about 1, wasaround 60-65 mg/mL.

For Samples 3-6, it can be seen that the difference between “Added SDEconcentration” and “Calculated SDE Concentration” was not significant,meaning the homogeneous formulations prepared by Method B were stableeven after being tested by three cycles of freeze-thaw step. The abovedata also demonstrate that when the weight ratio of benzyl benzoate tosesame oil was in the range of about 0.8-1.2:1, the present formulationsprepared by Method B can be homogeneous and stable with an SDEconcentration of 100 mg/mL or less. In some embodiments, the presentinvention may form a homogeneous and stable formulation, at the BB/oilratio of about 0.8-1.2:1, having a SDE concentration of 70-100 mg/mL,for example, 75 mg/mL. The SDE concentration of more than 70 mg/mLallows the injection volume to be greatly reduced, as compared with theinjection volume suggested by the prior art.

It should be understood that when the BB/oil ratio is increased, forexample, from about 1 to about 1.5, the solubility of SDE in the mixtureof BB and oil will be increased significantly, for example, from about60 mg/mL to about 150 mg/mL. Therefore, a homogeneously dissolvedformulation having a BB/oil ratio of about 1.5 can be prepared by eitherMethod A or Method B if the intended SDE concentration is lower than 150mg/mL. However, when a formulation having a BB/oil ratio of about 1.5needs to be prepared with a SDE concentration of greater than 150 mg/mL,Method B must be used to achieve a homogeneously dissolved formulation.

Example 3. In Vitro and In Vivo (on Dogs) Studies of PresentFormulations

(1) Preparation of Present Formulations

Three SDE formulations (AF3, AF1, and AF4) were prepared based on theconcentrations and BB/oil ratios listed in Table 3. AF4 was preparedaccording to Method A of Example 1; AF3 and AF1 were prepared accordingto Method B of Example 1.

TABLE 3 Present formulations for in vitro and in vivo studies on dogsSample Benzyl benzoate Sesame oil SDE BB/oil No. (w/w %) (w/w %) (mg/mL)ratio AF3 39 61 50 0.65 AF1 54.2 45.8 80 1.18 AF4 67 33 80 2

(2) Stability of Present Formulation

AF3, AF1 and AF4 formulations were then subject to a freeze-thaw test tocheck its physical stabilities. The freeze-thaw test was conducted bycooling each of the samples at about 0-4° C. for about 12 hours, warmingeach of the cooled samples at room temperature for about 12 hours, andsequentially repeating the cooling and warming steps twice. AF3, AF1,and AF4 formulations stayed clear and homogeneous after the freeze-thawtest. The resulting samples were centrifuged for 10 minutes at 3000 rpm.The upper solution of each sample was respectively collected and thensubjected to UPLC analysis according to the method of Example 1, exceptthat the run time was 15 minutes and the sample volume was 1 ul. “AddedSDE concentration” and “Calculated SDE Concentration” are the same asthat defined in Example 2, except that the upper solutions of AF3, AF1and AF4 were analyzed by UPLC rather than HPLC.

TABLE 4 UPLC analysis results of AF3, AF1 and AF4 formulations Added SDECalculated SDE Sample BB/oil concentration Concentration No. ratioAppearance (mg/mL) (mg/mL) AF3 0.65:1 Clear solution 50 47 AF1 1.18:1Clear solution 80 77 AF4   2:1 Clear solution 80 79

In Table 4, it can be seen that the difference between “Added SDEconcentration” and “Calculated SDE Concentration” is not significant,meaning the 3 formulations are homogeneous, and are stable even afterbeing subjected to three freeze-thaw cycles. The three formulations arerespectively prepared by Method A or Method B to form a homogeneoussolution without solid particles, and the homogeneous solutions exhibitsuperior stability that can satisfy commercial needs, such as beingsuitable for direct sterilization by filtration and low temperaturestorage (shelf-life may be prolonged when being stored at a lowertemperature).

(3) In Vitro Dissolution Experiment

In vitro dissolution experiments and UPLC analyses were performed,according to the processes disclosed in Example 1, on AF3, AF1 and AF4formulations. The accumulated SDE dissolution profiles of each of theformulations are plotted in FIG. 2. It can be seen from FIG. 2 that,when the BB/oil ratio is about 0.65, the time required for releasing 50%of the total amount of SDE from AF3 formulation into the in vitro mediumwas about 20-30 minutes. When the BB/oil ratio was about 1.18, the timerequired for releasing 50% of the total amount of SDE from AF1formulation into the in vitro medium was about 35-45 minutes. When theBB/oil ratio was about 2, the time required for releasing 50% of thetotal amount of SDE from AF4 formulation into the in vitro medium wasabout 50-60 minutes. This testing indicates that when the BB/oil ratiois increased from 0.65 to 2, the in vitro dissolution rate of thepresent formulation will be decreased.

(4) Determining the Effective Plasma Nalbuphine Concentration forAnalgesia in Dogs

In the Handbook of Veterinary Pain Management (2nd Edition, 2009), thepotencies of nalbuphine and other opioid analgesics were reported (page167, Table 9-2). The duration of analgesia when administratingnalbuphine at 0.5 mg/kg on dogs was reported to be about 4 hours. Thus,the plasma concentration of nalbuphine at 4 hours after injection of thesaid dose of nalbuphine via subcutaneous administration may be thelowest effective plasma concentration of nalbuphine for analgesia ondogs. According to in vivo experiments conducted by the inventors, theaverage plasma concentrations of nalbuphine at 3 hours and 4 hours aftersubcutaneously injected with 0.5 mg/kg nalbuphine were 9.9 ng/mL and 5.2ng/mL, respectively. Therefore, it may be regarded that the effectiveplasma concentration of nalbuphine for analgesia in dog is about 5ng/mL.

(5) Intramuscular Administration of Present Formulations into Dogs

An animal study was conducted to verify the correlation between the invitro dissolution rate and the in vivo release rate/release period ofthe present formulations. Each of the AF3, AF1, and AF4 formulationswere separately administered to two male Beagle dogs by intramuscularinjection. The dose of SDE was 160 mg for each dog, and the injectionvolumes varied according to the drug concentration of each formulation(AF3: 3.2 mL, AF1 and AF4: 2 mL). The blood samples were drawn prior tothe dosing, and at 1, 2, 6, 24, 36, 48, 60, 72, 96, 120 and 144 hoursafter dosing. The plasma nalbuphine concentration of each sample wasdetermined using a liquid chromatography-mass spectroscopy/massspectroscopy (LC-MS/MS) system consisting of an AB AP14000triple-quadrupole mass spectrometer coupled with Shimazdu LC-20AD and aCTC AutoSampler.

The mean plasma concentration-time profiles of nalbuphine from Day 1 toDay 6 for the three formulations are showed in FIG. 3, and thecorresponding log concentration-time profiles are shown in FIG. 4. SinceSDE is rapidly converted into nalbuphine when released from theformulation into the blood or tissues, the plasma nalbuphineconcentration is measured to represent the release amount of SDE fromthe formulation.

The in vivo testing results of the AF3, AF1 and AF4 formulations on dogsare consistent with the trend we expect based on the in vitrodissolution studies. It can be seen from FIG. 3 that when the BB/oilratio is increased, the in vivo release period of the formulation isextended or prolonged. More specifically, the nalbuphine plasmaconcentrations at 144 hours (Day 6) after administration of AF3, AF1 andAF4 are about 0.4 ng/mL, about 9.2 ng/mL and about 14.7 ng/mL,respectively. Based on an assumption that the effective plasmaconcentration of nalbuphine for analgesia on dog is about 5 ng/mL, AF3formulation would not produce an analgesic effect at 144 hours fromdosing, while the AF1 and AF4 formulations would produce an analgesiceffect for a period longer than 144 hours from dosing. According to theresult of dog experiment, when the BB/oil ratio was adjusted to lowerthan about 1, e.g. 0.65, the formulation may have a duration of actionof less than 6 days, for example, about 4 days; when the BB/oil ratiowas adjusted to greater than about 1, e.g. 2, the formulation may have aduration of action of equal to or greater than about 5, for example,more than 6 days. Thus, an extended release formulation of SDE with apredetermined release period can be prepared by adjusting the BB/oilratio of the formulation.

Furthermore, it can also be observed from FIG. 3 that when the BB/oilratio is increased, the release profile of the formulation will besteadier, e.g. delayed onset and insignificant burst release for AF4.The pharmacokinetic parameters are summarized in Table 5. The T_(max) ofAF3, AF1 and AF4 are 21 hours, 24 hours and 84 hours, respectively. TheC_(max) of AF3, AF1 and AF4 are 87.35 ng/mL, 82.90 ng/mL and 41.1 ng/mL,respectively.

When the BB/oil ratios were about 0.65, about 1, and about 2, the bloodconcentrations of nalbuphine at 1 hour after administration were about28.4 ng/mL, about 11.0 ng/mL, and about 6.7 ng/mL, respectively. Theresults indicate that when the BB/oil ratio is increased, the time toreach an effective blood concentration of nalbuphine may be longer, thusthe onset of action of the formulation may be slower.

The following PK parameters support the conclusion that when the BB/oilratio is increased, the formulation may exhibit a release profile with alonger release period, a lower maximum concentration (C_(max)) and alonger time to peak blood concentration level (T_(max)), as comparedwith a formulation having a lower BB/oil ratio.

TABLE 5 Pharmacokinetic parameters after IM injection of AF3, AF1 andAF4 formulations Formulation AF3 AF1 AF4 Injection volume (mL) 3.2 2 2Average BW(kg) 8.85 9.29 7.9 Average Dose(mg/kg) 18.13 17.24 20.2T_(max)(hr) 21.00 24.00 84.0 C_(max)(ng/mL) 87.35 82.90 41.1 T_(1/2)12.69 41.14 38.99 AUC_(last) (hr*ng/mL) 4555.63 5030.93 4075.7 AUC_(INF)_(—) _(pred) (hr*ng/mL) 4563.35 5577.12 4978.4

By using the Wegner-Nelson method, the average plasma concentrationversus time curve was transformed into the cumulative relative fractionabsorbed (CRFA) versus time curve. Under the assumption that Ab^(∞)(total amount of drug absorbed at t=0˜∞) of each formulation is 100% andthe elimination half-life of nalbuphine after i.v. dosing in dogs isabout 1.2 hour (Biopharm Drug Dispos. 1985 October-December;6(4):413-21.), the CRFA versus time curve of each formulation is plottedin FIG. 5. In FIG. 5, when the BB/oil ratio is about 0.65, about 99% ofAb^(∞) is absorbed at 144 hours after administration (AF3); when theBB/oil ratio is about 1.18, about 91% of Ab^(∞) is absorbed at 144 hoursafter administration (AF1); when the BB/oil ratio is about 2, about 82%of Ab^(∞) is absorbed at 144 hours after administration (AF4). Byapplying this model, it can be projected that when the BB/oil ratio ofthe formulation is increased, the relative percentage of Ab^(∞) absorbedat the same time point will be lower, which indicates that theformulation with a higher BB/oil ratio may release the dose depot for alonger period of time.

The above experimental results all indicate that the in vitrodissolution profiles of the present formulations are correlated to thein vivo release profiles of the formulations. The in vivo releaserate/release period of the present formulation can be controlled orregulated by the BB/oil ratio.

Example 4. In Vitro and In Vivo (on Humans) Studies of PresentFormulations

(1) Preparation of Present Formulations, by Method B of Example 1

About 600 g of SDE was mixed with about 4025 g of benzyl benzoate. Theresulting mixture was stirred at 300 rpm for 60 minutes to give a clearsolution. About 3591 g of sesame oil was added into the clear solution,and then stirred at 300 rpm for about 30 minutes. The resulting solutionwas subjected to filtration sterilization by using Millipore 0.22 μmfilters. The final formulation (F8) had an SDE concentration of about 75mg/mL, and the weight ratio of benzyl benzoate to sesame oil was about1.12:1 (Table 6).

TABLE 6 F8 formulation Sample Benzyl benzoate Sesame oil SDE BB/oil No.(w/w %) (w/w %) (mg/mL) ratio F8 53 47 75 1.12

The F8 formulation thus obtained was a homogeneous solution withoutsolid particles, thereby suitable for being sterilized directly byfiltration and suitable for large scale production.

(2) In Vitro Dissolution Experiment

In vitro dissolution experiments and UPLC analyses were performed,according to processes disclosed in Example 1, on the F8 formulation.The accumulated SDE dissolution profiles of the F8 formulation and theAF1 formulation are plotted in FIG. 6. It can be seen from FIG. 6 thatthe dissolution profiles of F8 and AF1 formulations are similar as theirBB/oil ratios are similar. When the BB/oil ratio is about 1.1-1.2, thetime required for releasing 50% of the total amount of SDE from theformulation into the in vitro medium was about 30-40 minutes.

In view of the duration of action results obtained with AF1 in dogsdescribed above and the similar in vitro dissolution profiles for F8 andAF1, a similar duration of action (i.e. greater than 6 days) with F8 wasexpected humans.

(3) Phase 1 Clinical Study

A Phase I clinical trial was conducted with the F8 formulation inhealthy volunteers. It was designed to evaluate safety and localtolerability and assess the pharmacokinetics of SDE following singledose intramuscular injection. The study enrolled a total of 28 healthymale subjects. All subjects were randomized to 5 cohorts. Cohort 1 (N=4)was treated with single dose Nubain® 17 mg (0.85 mL) via intramuscularinjection. Cohorts 2-5 (N=6) were either treated with placebo (N=2) ortreated with single dose SDE (N=4) at 75 mg (1 mL), 100 mg (1.33 mL),125 mg (1.67 mL), and 150 mg (2 mL), respectively. Overall, theescalating doses of SDE up to 150 mg were well tolerated. All adverseevents (AEs) were mild. No significant difference was found among theSDE and placebo groups in the number of AEs, the number of subjects withAEs, the severity of AEs, and AE relatedness.

Blood was collected from all 28 subjects who were treated with Nubain®,the F8 formulation, or placebo. Heparinized blood samples were obtainedbefore and at various time points following single dose administrationof study medication. Nalbuphine from Nubain® or the F8 formulation weredetected and quantitated using the validated LC/MS/MS method. Thesubsequent analysis of the data involved non-compartmentalpharmacokinetics analysis, i.e., C_(max), T_(max), AUC_(0-t),AUC_(0-int), and T_(1/2). Since SDE is rapidly converted to nalbuphinein the blood, the pharmacokinetic parameters of nalbuphine werecalculated using a non-compartment model and actual time vs. plasmaconcentrations of nalbuphine (Table 7).

The pharmacokinetics of nalbuphine following the administration of theF8 formulation appeared to be dose-proportional. The highest meanC_(max) and AUC_(0-int) were found at 150 mg SDE and were 9.81 ng/mL and1353.16 ng*hr/mL, respectively. Plasma nalbuphine reached C_(max) within45-66 hours (T_(max)) after IM administration of 75, 100, 125, and 150mg SDE.

The average elimination half-life (T₁/2) of nalbuphine after IMadministration of Nubain® (cohort 1) was about 4 h. Followingintramuscular injection of the F8 formulation with a total dose rangingfrom 75 mg to 150 mg, the mean apparent T_(1/2) of nalbuphine rangedfrom about 56 to 90 hours. The longer apparent half-life was most likelydue to slow and prolonged absorption of SDE/nalbuphine from the IMinjection site.

TABLE 7 Pharmacokinetic parameters after single IM injection of 17 mgNubain ® (NH) or escalating doses of F8 formulation. Treat- ment/C_(max) T_(max) AUC_(0-t) AUC_(0-inf) T_(1/2) Dose (ng/mL) (hr)(hr*ng/mL) (hr*ng/mL) (hr) NH Mean 85.73 0.25 171.74 174.16 4.06  17 mgSD 18.61 0 15.99 15.53 1.13 SDE Mean 5.45 66 647.84 692.27 55.75  75 mgSD 0.85 22.98 96.95 114.00 5.54 SDE Mean 5.65 66 811.34 936.17 85.45 100mg SD 1.50 40.99 145.69 176.27 29.70 SDE Mean 7.31 45 1047.45 1219.7189.83 125 mg SD 1.79 24.74 205.77 263.16 24.74 SDE Mean 9.81 60 1214.981353.16 73.4 150 mg SD 2.94 13.86 155.93 137.55 30.74

FIG. 7 shows the plasma concentrations of nalbuphine followingadministration of various dose of the F8 formulation and of one dose ofNubain®. The plasma concentrations of nalbuphine were above 1 ng/mL or 3ng/mL for at least 220 or 120 hours, respectively, in all 16 subjectsreceiving 75-150 mg SDE in the F8 formulation. However, followingintramuscular injection of Nubain® 17 mg, the plasma concentrations ofnalbuphine remained above 1 ng/mL or 3 ng/mL for only 16 or 8 hours,respectively. Under the assumption that the effective plasmaconcentration of nalbuphine for analgesia in moderate to severe pain isgreater than 1 ng/mL, more preferably greater than 3 ng/mL (Can JAnaesth. 1991 March; 38(2): 175-82; European Journal of ClinicalPharmacology 1987, Volume 33, Issue 3, pp 297-301), the duration ofaction of a single injection of the F8 formulation would be much longerthan that covered by a single injection of Nubain®. In the cohortreceiving 125 mg SDE, the mean plasma concentration of nalbuphine wasabove 3 ng/mL between 12 to 168 hours of dosing, which indicated thatthe duration of action was about 6.5 days (168−12=156 hours=6.5 days).In the cohort receiving 150 mg SDE, the mean plasma concentration ofnalbuphine was above 3 ng/mL between 24 to 168 hours of dosing, and wasabove 1 ng/mL between 6 to 288 hours of dosing, which indicated that theduration of action was about 6-12 days (168−24=144 hours=6 days;288−6=282 hours=11.75 days). For some individuals, the plasmaconcentrations of nalbuphine were above 3 ng/mL between 12 to 216 hoursof dosing, which indicated that the duration of action was about 8.5days (216−12=204 hours=8.5 days). This indicates that the F8 formulationmay be administered to a patient at 6 to 36 hours prior to the onset ofpain symptoms. For example, the onset of pain symptoms is during orafter a surgical operation. Accordingly, the F8 formulation may beadministered to a patient at 6 to 36 hours prior to a surgicaloperation, and may effectively relieve pain during and immediately afterthe surgical operation. For example, the F8 formulation may beadministered at 12-36 hours or 12-24 hours prior to the surgicaloperation.

(4) Bioavailability Study

A bioavailability study with the F8 formulation and Bain® (NalbuphineHCl IM injection) was conducted on healthy volunteers. A total of twelvesubjects completed the crossover study. Each subject received a singledose of reference drug (Bain®, Nalbuphine HCl IM injection, 10 mg/mL×2mL) in period I and the F8 formulation (SDE IM injection, 75 mg/mL×2 mL)in period II. There was a minimum 5-day washout period between period Iand period II. In period I, the blood samples were drawn prior to thedosing, and 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hoursafter dosing. In period II, the blood samples were drawn prior to thedosing, and 6, 12, 24, 48, 60, 72, 96, 120, 168, 216, 288 and 360 hoursafter dosing.

The whole blood concentrations of nalbuphine in the samples weredetermined by LC-MS/MS. AUC_(0-t), AUC_(0-inf), C_(max), T_(max),T_(1/2), and MRT for nalbuphine in whole blood were determined bynon-compartment methods. In addition, relative bioavailability of SDEcompared to Nalbuphine (Bain®) was calculated.

No serious adverse events occurred during this study. Pharmacokineticparameters of nalbuphine for Bain® and the F8 formulation are presentedas mean±the standard deviation (SD) in Table 8.

TABLE 8 Pharmacokinetic parameters of nalbuphine for Nalbuphine (Bain ®)and the F8 formulation. Treat- ment/ AUC_(0-t) AUC_(0-inf) C_(max)T_(max) MRT T_(1/2) Dose (ng/mL*h) (ng/mL*h) (ng/mL) (h) (h) (h) Bain ®Mean 319.3 323.0 115.45 0.40 4.15 4.03  20 mg SD 85.3 86.5 56.45 0.130.61 0.54 SDE Mean 1687.4 1832.0 15.40 64.00 149.38 83.16 150 mg SD385.6 402.2 6.42 9.34 69.07 46.45

FIG. 8 shows the mean whole blood concentration-time profiles ofnalbuphine after subjects received the F8 formulation from 0 to 360hours of dosing in period II described above. In this bioavailabilitystudy, the mean whole blood concentration of nalbuphine was above 3ng/mL between 24 to 168 hours of dosing, which indicated that theduration of action was about 6 days (Table 9). The result is consistentwith the finding in the cohort receiving 150 mg SDE of Phase 1 study(plasma concentration of nalbuphine). For some individuals, the wholeblood concentrations of nalbuphine were above 3 ng/mL between 12 to 216hours of dosing, which indicated that the duration of action was about8.5 days. In addition, the mean whole blood concentration of nalbuphinewas above 1 ng/mL between 6 to 288 hours of dosing, which indicated thatthe duration of action was about 12 days.

TABLE 9 Mean whole blood concentration-time data of nalbuphine for theF8 formulation Time Mean (h) (ng/mL) SD 0 — — 6 1.09 0.39 12 2.04 0.9424 5.03 2.55 48 11.83 5.51 60 13.39 5.87 72 14.12 6.49 96 9.17 3.15 1206.48 1.67 168 4.33 1.19 216 2.51 1.10 288 1.44 0.98 360 0.81 0.78

The relative bioavailability (F) of nalbuphine from the F8 formulationto nalbuphine from Bain® was about 86.2±12.1(%). In addition, the meanabsorption time (MAT) and absorption rate (Ka) of nalbuphine from the F8formulation were about 145.2±69.1 hour and about 0.0081±0.0030 h⁻¹,respectively. The bioavailability study shows that the duration ofaction of the present formulation may be equal to or greater than 6 dayswhen the BB/oil ratio is set to about 1.12. On some individuals, theduration of action of the present formulation may be about 5 days.

By correlating the dog study results (Example 3) with the human studyresults, it can be concluded that when the formulation having a BB/oilratio of less than 1, e.g., about 0.65, is administered to a humansubject via IM injection, the duration of action of the formulation maybe less than 6 days; while when the formulation having a BB/oil ratio ofmore than 1, e.g., about 2, is administered to a human subject via IMinjection, the duration of action of the formulation may be greater than6 days. Accordingly, the present invention may provide extended releaseformulations of SDE with various release periods by adjusting the BB/oilratio. For example, when a longer duration of action is intended (e.g. 1or 2 weeks, or longer), the BB/oil ratio may be set to about 1 orgreater than 1, for example, 2 or 3; and when a shorter duration ofaction is intended (e.g. 3 or 4 days), the BB/oil ratio may be set toless than 1, for example, 0.5 or 0.65.

(5) Phase 2/3 Clinical Study

A random, placebo-controlled, single dose, parallel design, phase 2/3study was conducted with 209 male and female patients to assess thesafety and efficacy of the intramuscular injection of F8 formulation inthe treatment of postoperative pain following hemorrhoidectomy.

The subjects were divided into two groups, of which Group 1 (n=103) wastreated with a single dose of intramuscular SDE 150 mg (2 mL), and Group2 (n=106) was treated with a single dose of intramuscular placebo (2mL). All subjects were given a single dose of SDE or placebo viaintramuscular injection 24±12 hours before hemorrhoid surgery. Thesubjects were allowed to take rescue medication and monitored for 7 daysafter dosing. Statistical analyses were performed on the data to comparethe two groups.

The primary efficacy endpoint was pain assessment calculated as the areaunder the curve of VAS pain intensity scores through 48 hours aftersurgery. The secondary efficacy endpoints included pain assessmentmeasured with VAS; time from the end of operation to the first rescuemedication dosing; the consumption of oral ketorolac. Pain intensity wasassessed right before the first use of PCA ketorolac, and at 1±0.1,20.1, 30.1, 4±0.25, 8±0.5, 12±0.5, 16±0.5, 20±0.5, 24±1, 28±1, 32±2,36±2, 40±2, 44±2, 48±2 hours after the surgery, and was assessed duringDays 3-7 in the morning and evening, as well as during special eventssuch as bowel movements.

For the calculation of AUC, data were imputed with the use of thewindowed worst observation carried forward plus last observation carriedforward method. For subjects who used rescue medication for pain relief,their VAS scores recorded within the window of ketorolac medication (6hours, which is one half-life of ketorolac) were replaced by the “worst”observation (i.e., the highest score before taking ketorolac), hereaftercalled “adjusted VAS scores.”

The AUC₀₋₂₄ and AUC₀₋₄₈ of the mean adjusted VAS scores of SDE andplacebo groups were calculated by using the trapezoidal method andsummarized in Table 10.

TABLE 10 Statistical analysis of AUC₀₋₂₄ and AUC₀₋₄₈ by treatment afterhemorrhoid operation. Mean ± SD SDE − Placebo Population SDE PlaceboLS-mean [95% CI]¹ p-value² mITT N 103 106 AUC₀₋₂₄ 109.42 ± 55.04  126.71± 49.22  −16.86 [−31.05; −2.67]  0.0201* AUC₀₋₄₈ 209.93 ± 111.26 253.53± 108.49 −42.20 [−71,68; −12.71] 0.0052* *mITT population: modifiedIntention-To-Treat population

The AUC₀₋₄₈ of the SDE group showed statistically significantsuperiority against placebo group in mITT population (209.93±111.26 vs.253.53±108.49, p=0.0052). The distribution statistics for the amount oforal ketorolac consumption after 48 hours post-operation by treatmentwas graphed as the box plot presented in FIG. 9. Both the mean andmedian consumption of oral ketorolac were lower in SDE group (Mean:51.36 mg, Median: 40.00 mg) than those in the placebo group (Mean: 73.30mg, Median: 80.00 mg).

Furthermore, the time of the first use of post-surgical analgesic frompost-operation was assessed and the results of distribution statisticsare summarized in Table 11. Longer periods of time for the first use ofpost-operational analgesic in SDE groups were observed. The mean timeperiod of SDE group (12.57 hours) was prolonged, compared to that of theplacebo group (4.93 hours).

TABLE 11 Distribution statistics of time (hour) for the first use ofpost-operational analgesic by treatment Population SDE Placebo OverallmITT (N) 103 106 209 NObs¹ 87 99 186 Mean (SD) 12.57 (1.68) 4.93 (0.47)9.68 (1.01) Q1 [95% CI]² 2.27 [1.32; 2.78] 1.43 [0.98; 1.90] 1.80 [1.23;2.22] Median [95% CI]^(2, 3) 4.42 [3.47; 5.80] 3.28 [2.33; 4.73] 4.23[3.03; 4.75] Q3 [95% CI]² 11.25 [8.33; 45.65] 6.52 [5.67; 7.60]  7.95[6.60; 10.63]

In summary, the trends of VAS scores for pain intensity measured overtime through 48 hours post-operation, the time from the end of operationto the first rescue medication dosing and the consumption of oralketorolac within 48 hours were consistent. All these clinical resultsindicated that the F8 formulation may be administered to a patient priorto a hemorrhoid operation, and may effectively relieve pain immediatelyafter the hemorrhoid operation.

FIG. 10 shows the plot of adjusted VAS scores over time through 0 hr to7 days after hemorrhoid operation of mITT population. The adjustedAUC_(0-final) of the mean VAS scores for SDE and placebo groups werecalculated by using the trapezoidal method and summarized in Table 12.The AUC_(0-final) of mean adjusted VAS scores of pain intensity of theSDE group showed statistically significant superiority against theplacebo group in mITT population (630.79±350.90 vs. 749.94±353.72,p=0.0165). In addition, the adjusted VAS score of pain intensity in theSDE group was lower at beginning of the assessment measured the first 1hour and at Day 7 morning after hemorrhoid operation (see the first andthe last data point of FIG. 10). The adjusted VAS score of the SDE groupwas lower than the placebo group throughout the 7 days after thehemorrhoid operation. Accordingly, the F8 formulation may beadministered to a patient prior to a hemorrhoid operation, and mayeffectively relieve pain immediately after the hemorrhoid operation withthe duration of action persisting for at least about 5 or 6 days.

TABLE 12 Statistical analysis of AUC_(0-final) (based on adjusted VASscores) by treatment after hemorrhoid operation Mean ± SD SDE − PlaceboPopulation SDE Placebo LS-mean [95% CI]¹ p-value² mITT N 103 106AUC_(0-final) 630.79 ± 350.90 749.94 ± 353.72 −115.20 [−209.153; −21.26]0.0165*

Example 5. Stability Data of the Present Formulation

The F8 formulation prepared according to Example 4 was stored at 2-8° C.for 24 months. The formulation remained as a clear and lightly yellowoily solution for the whole period of 24 months. In the meantime, theformulation was tested periodically by HPLC to determine its Assay(Table 13).

TABLE 13 Assay of the F8 formulation at 2-8° C. for 24 months. Time ofsampling Assay by HPLC (month) (% of target) 0 104.3% 6 105.2% 12 101.7%18 101.7% 24 101.3%

It can be seen from Table 13 that the F8 formulation is stable forstorage at 2-8° C. for at least 24 months. The freeze-thaw testconducted in Example 2 also demonstrates that the formulation of thepresent invention can remain homogeneous at about 0-4° C. while notforming precipitates or solid particles when being returned to roomtemperature. The stability testing results indicate that theformulation, having a SDE concentration (about 75 mg/mL) higher than theoriginal solubility/saturated concentration (about 60 mg/mL), preparedby Method B can exhibit superior stability to satisfy commercial needs,e.g. long shelf-life under low-temperature storage.

In another example, the F8 formulation was respectively stored at 5, 25and 40° C. for 6 months. Samples collected from each group were testedperiodically by HPLC to determine the formation of degradation products(Table 14).

TABLE 14 Stability of the F8 formulation at 5, 25, and 40° C. for 6months Time of sampling Total impurities by HPLC (month) Stored at 5° C.Stored at 25° C. Stored at 40° C. 0 0.37% 0.37% 0.37% 1 0.42% 0.45%0.47% 3 0.39% 0.50% 0.85% 6 0.38% 0.65% 1.66%

It can be seen from Table 14 that when stored at 25° C. or 40 OC, the F8formulation generated more impurities. This indicates that theformulation is more suitable to be stored at a temperature lower thanroom temperature. Since the formulation of the present invention canremain homogeneous and stable at 2-8° C. while not forming solidparticles when returned to room temperature, its shelf-life can befurther prolonged by being stored at a lower temperature.

Example 6. Improvement of SDE Solubility in Various Solvent SystemsContaining Different Alkyl Alcohols as the Solubilizing Agent

(1) Solubility Test of SDE Solutions

Table 15 shows the compositions of the tested solvent systems byweight/weight percent (w/w %). The solvent systems each comprise sesameoil, benzyl benzoate and different alkyl alcohols (i.e., ethanol,1-propanol, 2-propanol, 1-butanol and t-butanol).

Each of the solvent systems was prepared by mixing each component withthe exact volume (μl) corresponding to the w/w % listed in Table 15.About 500 μl of each of the solvent systems was prepared and wasvortexed for 3 minutes or more to fully mix each component. Anappropriate amount of SDE was added into 250 μl of each of the solventsystems and the resulting mixtures were sonicated for 10 minutes. If thepreviously added SDE was completely dissolved, an additional 4-8 mg ofSDE was added into the mixture and which was then sonicated for another10 minutes. The step of adding 4-8 mg of SDE was repeated until themixture was saturated to show undissolved SDE precipitates. All of themixtures were sonicated for at least 30 minutes, and then centrifuged tocollect the supernatants. The supernatants were treated withacetonitrile and then subjected to UPLC analysis.

(2) UPLC Analysis

The SDE concentrations in the supernatants collected from samples S1-S8were respectively determined by UPLC according to the method of Example1, except that the run time was 15 minutes and the sample volume was 1ul.

The saturated SDE concentrations (i.e., solubility) for each testedsolvent systems are shown in Table 15. It is seen that the addition ofalkyl alcohols in the solvent systems can significantly increase the SDEsolubility, although the solubility of SDE in each of the alcohols aloneis rather low. The solubility of SDE in ethanol, 1-propanol, ort-butanol is about 10-30 mg/mL (Table 16).

The solubility of SDE in the solvent systems may be increased by atleast 30% when there is 10% of alkyl alcohol added.

TABLE 15 Effect of alkyl alcohol on increasing SDE solubility in thepresent formulations Composition of the solvent system (w/w %) SDESolubility alcohol sesame benzyl Solubility Improve- No. type alcoholoil benzoate (mg/mL) ment S1 NC* 0 40 60 159.8 — S2 Ethanol 10 40 50267.7 168% S3 1- 10 40 50 254.1 159% Propanol S4 2- 10 40 50 235.2 147%Propanol S5 1- 10 40 50 222.0 139% Butanol S6 tert- 10 40 50 211.2 132%Butanol *NC: Negative control, i.e. no alcohol is added.

TABLE 16 The solubility of SDE in various solvents Sample Solubility ofSDE in the solvent No. Solvent (mg/mL) 1 Benzyl benzoate (BB) 373.44 2Benzyl alcohol 583.13 4 Ethanol 15.86 5 1-Propanol 32.46 6 t-Butanol19.46 11 Sesame oil 5.93

Example 7. Solubility of SDE in Solvent Systems Comprising DifferentTypes and Amounts of Alkyl Alcohols

Five groups of solvent systems containing 40% by weight of sesame oilwere respectively prepared according to the method of Example 6 by usingthe compositions as listed in Table 17; and among them, five differentalcohols (i.e., ethanol, 1-propanol, 2-propanol, 1-butanol, andtert-butanol) were used in the five groups. SDE was dissolved in each ofthe solvent systems till saturation by following the method of Example6. The SDE concentrations in the supernatants collected from each of thesamples (i.e. the saturated SDE concentration, or SDE solubility) wererespectively determined by UPLC using the method of Example 6.

TABLE 17 Solvent systems with various types and amounts of alcoholsComponents of the solvent system (w/w %) Sesame oil Alcohol * Benzylbenzoate 40 0 60 40 2.5 57.5 40 5 55 40 10 50 40 20 40 40 30 30 40 4020 * Ethanol, 1-propanol, 2-propanol, 1-butanol, and tert-butanol wereused in the five groups, respectively.

The UPLC analysis results are divided into the five groups to illustratethe effect of changing the alcohol on the SDE solubility, as depicted inFIG. 11. It can be seen from FIG. 11 that when ethanol is replaced by1-propanol, 2-propanol, 1-butanol, or tert-butanol, the trends ofsolubility improvement observed are similar. For C₂-C₄ alcohols, when2.5-30% by weight of the alcohol is added into the solvent system, thesolubility of SDE can be significantly increased; and when about 10-20%by weight of the alcohol is added into the solvent system, the SDEsolubility can be maximized.

Example 8. In Vitro Release Study of Present Formulations ContainingVarious Alkyl Alcohols

(1) Preparation of Present Formulations

Five present formulations were prepared according to Method A of Example1 (i.e., mixing the alkyl alcohol, BB and oil, then adding SDE) by usingvarious solvent systems as listed in Tables 18. The solubility of SDE informulations comprising an oli-miscible retaining solvent and apharmaceutically acceptable oil may be significantly increased by addingan alkyl alcohol.. As such, Method A is sufficient to prepare ahomogenous alcohol-containing formulation having a SDE concentrationthat is greater than the SDE solubility of the formulation without thealcohol, as long as the intended concentration of SDE in thealcohol-containing formulation is lower than the saturatedconcentration.

For the five formulations listed in Table 18, each formulation contains10% ethanol and the BB/oil ratio ranges from 0.5 to 16.

TABLE 18 The present formulations with ethanol, at various BB/oil ratiosSample Ethanol Benzyl benzoate Sesame oil SDE BB/oil No. (w/w %) (w/w %)(w/w %) (mg/mL) ratio N1 10 30 60 75 0.5 N2 10 45 45 150 1 N3 10 67.522.5 150 3 N4 10 80 10 150 8 N5 10 85 5 150 16

(2) In Vitro Dissolution Experiment

In vitro dissolution experiments and UPLC analyses were performedaccording to the method of Example 1. The accumulated SDE dissolutionprofiles of the five formulations listed in Tables 18 are plotted inFIG. 12.

In FIG. 12, it can be seen that the trend observed in FIG. 1 remainswhen ethanol is added into the formulations (N1-N5 of Table 18).

The presence or absence of an alkyl alcohol in the present formulationdoes not significantly affect the correlation between thedissolution/release rate and the BB/oil ratio, though the addition ofthe alcohol can significantly improve the solubility of SDE in theformulation so as to give a homogeneous formulation having a SDEconcentration higher than that possible for the formulation without thealcohol.

Example 9. In Vitro Release Study of Present Formulations with orwithout Alkyl Alcohol

(1) Preparation of Formulations

Six formulations were prepared using various solvent systems as listedin Tables 19-20. Among them, formulations F8 and AF1 were preparedaccording Method B of Example 1, and the remaining formulations wereprepared according to Method A of Example 1 (i.e. mixing the alkylalcohol, BB and oil, then adding SDE).

For the three formulations listed in Table 19, the BB/oil ratios are all16. For the N5 and N10 formulations, ethanol and 1-butanol wererespectively added in the formulations. For the N7 formulation, therewas no alcohol used in the formulation.

For the three formulations listed in Table 20, the BB/oil ratios are allabout 1. For the N2 formulation, ethanol was added in the formulation;and for the F8 and AF1 formulations, there were no alcohol used in theformulations.

TABLE 19 The present formulations with/without alcohols, at the BB/oilratio of 16 Components of the solvent system (w/w %) Sample AlcoholBenzyl Sesame SDE BB/oil No. type Alcohol benzoate oil (mg/mL) ratio N7— 0 94 6 150 16 N5 Ethanol 10 85 5 150 16 N10 1-butanol 10 85 5 150 16

TABLE 20 The present formulations with/without alcohols, at the BB/oilratio of about 1 Components of the solvent system (w/w %) Sample BenzylSesame SDE BB/oil No. Ethanol benzoate oil (mg/mL) ratio F8 0 53 47 751.12 AF1 0 54.2 45.8 80 1.18 N2 10 45 45 150 1

(2) In Vitro Dissolution Experiment

In vitro dissolution experiments and UPLC analyses were performed,according to the method of Example 1, on the formulations listed inTables 19-20. The accumulated SDE dissolution profiles of the eightformulations of Tables 19 and 20 are plotted in FIGS. 13-14,respectively. As shown in FIGS. 13-14, the presence or absence of analkyl alcohol, does not significantly affect the dissolution rate of SDEfrom the formulation as long as the BB/oil ratio remains the same orsimilar. When the BB/oil ratio is set to about 16, the times requiredfor releasing 50% of the total amount of SDE from the four formulationsinto the in vitro medium are all about 100-120 minutes (N7, N5 and N10of Table 19). When the BB/oil ratio is set to about 1, the timesrequired for releasing 50% of the total amount of SDE from the threeformulations into the in vitro medium are all about 20-50 minutes (F8,AF1, and N2 of Table 20).

The results show that the presence or absence of an alkyl alcohol doesnot significantly affect the dissolution/release rate of the presentformulations as long as the BB/oil ratio remains the same or similar.

Example 10. In Vitro Release Study of Formulations Containing DifferentOils

(1) Preparation of Formulations

Four formulations were prepared according to Method A of Example 1 byusing different solvent systems as listed in Table 21 (i.e., mixingethanol, BB and the oil, then adding SDE). For the N2 and N14formulations, the BB/oil ratios are about 1; while for the N4 and N13formulations, the BB/oil ratios are about 8. In the N2 and N4formulation, the oil used is sesame oil; and in the N14 and N13formulation, the oil used is castor oil.

TABLE 21 The formulations with different oils Components of the solventsystem (w/w %) Sample Oil Benzyl SDE BB/oil No. Type Oil Ethanolbenzoate (mg/mL) ratio N2 sesame oil 45 10 45 150 1 N14 castor oil 45 1045 150 1 N4 sesame oil 10 10 80 150 8 N13 castor oil 10 10 80 150 8

(2) In Vitro Dissolution Experiment

In vitro dissolution experiments and UPLC analyses were performed,according to the method in Example 1, on the four formulations. Theaccumulated SDE dissolution profiles of the four formulations areplotted in FIG. 15.

As shown in FIG. 15, when the oil used is sesame oil and the BB/oilratio is set to 1, the time required for releasing about 50% of thetotal amount of SDE from the formulation into the in vitro medium isabout 30-40 minutes (N2); and when the oil used is castor oil and theBB/oil ratio is set to 1, the time required for releasing 50% of thetotal amount of SDE from the formulation into the in vitro medium isabout 100-120 minutes (N14). On the other hand, when the oil used issesame oil and the BB/oil ratio is set to 8, the time required forreleasing about 50% of the total amount of SDE from the formulation intothe in vitro medium is about 70-90 minutes (N4); and when the oil usedis castor oil and the BB/oil ratio is set to 8, the time required forreleasing 50% of the total amount of SDE from the formulation into thein vitro medium is about 120-140 minutes (N13).

It can be seen from FIG. 15 that the dissolution profiles of the N2 andN14 formulations are quite similar though the dissolution rate can befurther decreased by using castor oil to replace sesame oil. Thispattern is also seen with the comparison between N4 and N13formulations. This is to say that to replace sesame oil by anotherpharmaceutical acceptable oil in the present formulation will notsignificantly affect the correlation between the release period and theBB/oil ratio, i.e., the higher BB/oil ratio, the longer the releaseperiod of the formulation. Furthermore, using castor oil to replace withsesame oil may further prolong the release period of the formulation.

1-40. (canceled)
 41. An extended release pharmaceutical formulationcomprising sebacoyl dinalbuphine ester (SDE), benzyl benzoate, andsesame oil, wherein: the weight ratio of the benzyl benzoate to thesesame oil is about 1.1:1; the concentration of the SDE in theformulation is at least about 75 mg/mL; and the formulation is ahomogeneous solution substantially free of solid particles and suitablefor administration to a human subject by intramuscular injection of asingle dose having a volume of less than about 5 mL.
 42. The extendedrelease pharmaceutical formulation of claim 41, wherein the volume isabout 1 mL to about 4 mL.
 43. The extended release pharmaceuticalformulation of claim 42, wherein the volume is about 2 mL to about 4 mL.44. The extended release pharmaceutical formulation of claim 42, whereinthe volume is about 2 mL.
 45. The extended release pharmaceuticalformulation of claim 42, wherein the volume is about 3 mL.
 46. Theextended release pharmaceutical formulation of claim 42, wherein thevolume is about 4 mL.
 47. The extended release pharmaceuticalformulation of claim 41, wherein the concentration of the SDE in theformulation is about 75 mg/mL.
 48. The extended release pharmaceuticalformulation of claim 41, wherein the single dose comprises about 75 mgto about 300 mg of SDE.
 49. The extended release pharmaceuticalformulation of claim 48, wherein the single dose comprises about 150 mgto about 300 mg of SDE.
 50. The extended release pharmaceuticalformulation of claim 48, wherein the single dose comprises about 150 mgof SDE.
 51. The extended release pharmaceutical formulation of claim 48,wherein the single dose comprises about 225 mg of SDE.
 52. The extendedrelease pharmaceutical formulation of claim 48, wherein the single dosecomprises about 300 mg of SDE.
 53. The extended release pharmaceuticalformulation of claim 41, wherein the weight ratio of the benzyl benzoateto the sesame oil is about 1.12:1.
 54. The extended releasepharmaceutical formulation of claim 53, wherein the concentration of theSDE in the formulation is about 75 mg/mL.
 55. The extended releasepharmaceutical formulation of claim 41, wherein the formulation isprepared by: (1) dissolving the SDE in the benzyl benzoate; and (2)mixing the resulting solution with the sesame oil to obtain ahomogeneous solution.
 56. The extended release pharmaceuticalformulation of claim 55, wherein the homogeneous solution is sterilizedby filtration.
 57. The extended release pharmaceutical formulation ofclaim 41, wherein the formulation does not comprise precipitates orsolid particles.
 58. The extended release pharmaceutical formulation ofclaim 41, wherein the formulation remains homogeneous at 2-8° C. for atleast 6 months.
 59. The extended release pharmaceutical formulation ofclaim 58, wherein the formulation remains homogenous after one or morefreeze-thaw cycles.